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Sample records for reveal flexible domains

  1. Structure of a mutant [beta] toxin from Staphylococcus aureus reveals domain swapping and conformational flexibility

    SciTech Connect

    Kruse, Andrew C.; Huseby, Medora J.; Shi, Ke; Digre, Jeff; Ohlendorf, Douglas H.; Earhart, Cathleen A.

    2011-09-16

    The 3.35 {angstrom} resolution crystal structure of a mutant form of the staphylococcal sphingomyelinase {beta} toxin in which a conserved hydrophobic {beta}-hairpin has been deleted is reported. It is shown that this mutation induces domain swapping of a C-terminal {beta}-strand, leading to the formation of dimers linked by a conformationally flexible hinge region. Eight dimers are seen in the asymmetric unit, exhibiting a broad spectrum of conformations trapped in place by intermolecular contacts within the crystal lattice. Furthermore, the 16 monomers within each asymmetric unit exhibit a remarkable heterogeneity in thermal factors, which can be accounted for by the varying degrees to which each monomer interacts with other molecules in the crystal. This structure provides a unique example of the challenges associated with crystallographic study of flexible proteins.

  2. Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

    PubMed Central

    Weisshuhn, Philip C.; Sheppard, Devon; Taylor, Paul; Whiteman, Pat; Lea, Susan M.; Handford, Penny A.; Redfield, Christina

    2016-01-01

    Summary The Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4. PMID:26996961

  3. A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

    PubMed Central

    Zhao, Yongqian; Soh, Tingjin Sherryl; Zheng, Jie; Chan, Kitti Wing Ki; Phoo, Wint Wint; Lee, Chin Chin; Tay, Moon Y. F.; Swaminathan, Kunchithapadam; Cornvik, Tobias C.; Lim, Siew Pheng; Shi, Pei-Yong; Lescar, Julien; Vasudevan, Subhash G.; Luo, Dahai

    2015-01-01

    Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5. PMID:25775415

  4. Flexible time domain averaging technique

    NASA Astrophysics Data System (ADS)

    Zhao, Ming; Lin, Jing; Lei, Yaguo; Wang, Xiufeng

    2013-09-01

    Time domain averaging(TDA) is essentially a comb filter, it cannot extract the specified harmonics which may be caused by some faults, such as gear eccentric. Meanwhile, TDA always suffers from period cutting error(PCE) to different extent. Several improved TDA methods have been proposed, however they cannot completely eliminate the waveform reconstruction error caused by PCE. In order to overcome the shortcomings of conventional methods, a flexible time domain averaging(FTDA) technique is established, which adapts to the analyzed signal through adjusting each harmonic of the comb filter. In this technique, the explicit form of FTDA is first constructed by frequency domain sampling. Subsequently, chirp Z-transform(CZT) is employed in the algorithm of FTDA, which can improve the calculating efficiency significantly. Since the signal is reconstructed in the continuous time domain, there is no PCE in the FTDA. To validate the effectiveness of FTDA in the signal de-noising, interpolation and harmonic reconstruction, a simulated multi-components periodic signal that corrupted by noise is processed by FTDA. The simulation results show that the FTDA is capable of recovering the periodic components from the background noise effectively. Moreover, it can improve the signal-to-noise ratio by 7.9 dB compared with conventional ones. Experiments are also carried out on gearbox test rigs with chipped tooth and eccentricity gear, respectively. It is shown that the FTDA can identify the direction and severity of the eccentricity gear, and further enhances the amplitudes of impulses by 35%. The proposed technique not only solves the problem of PCE, but also provides a useful tool for the fault symptom extraction of rotating machinery.

  5. High-resolution cryo-electron microscopy structures of murine norovirus 1 and rabbit hemorrhagic disease virus reveal marked flexibility in the receptor binding domains.

    PubMed

    Katpally, Umesh; Voss, Neil R; Cavazza, Tommaso; Taube, Stefan; Rubin, John R; Young, Vivienne L; Stuckey, Jeanne; Ward, Vernon K; Virgin, Herbert W; Wobus, Christiane E; Smith, Thomas J

    2010-06-01

    Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to approximately 8-A resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.

  6. Single particle electron microscopy analysis of the bovine anion exchanger 1 reveals a flexible linker connecting the cytoplasmic and membrane domains.

    PubMed

    Jiang, Jiansen; Magilnick, Nathaniel; Tsirulnikov, Kirill; Abuladze, Natalia; Atanasov, Ivo; Ge, Peng; Narla, Mohandas; Pushkin, Alexander; Zhou, Z Hong; Kurtz, Ira

    2013-01-01

    Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO₂ transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.

  7. Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains

    PubMed Central

    Abuladze, Natalia; Atanasov, Ivo; Ge, Peng; Narla, Mohandas; Pushkin, Alexander; Zhou, Z. Hong; Kurtz, Ira

    2013-01-01

    Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity. PMID:23393575

  8. The frontal assessment battery (FAB) reveals neurocognitive dysfunction in substance-dependent individuals in distinct executive domains: Abstract reasoning, motor programming, and cognitive flexibility.

    PubMed

    Cunha, Paulo Jannuzzi; Nicastri, Sergio; de Andrade, Arthur Guerra; Bolla, Karen I

    2010-10-01

    Substance-dependence is highly associated with executive cognitive function (ECF) impairments. However, considering that it is difficult to assess ECF clinically, the aim of the present study was to examine the feasibility of a brief neuropsychological tool (the Frontal Assessment Battery - FAB) to detect specific ECF impairments in a sample of substance-dependent individuals (SDI). Sixty-two subjects participated in this study. Thirty DSM-IV-diagnosed SDI, after 2weeks of abstinence, and 32 healthy individuals (control group) were evaluated with FAB and other ECF-related tasks: digits forward (DF), digits backward (DB), Stroop Color Word Test (SCWT), and Wisconsin Card Sorting Test (WCST). SDI did not differ from the control group on sociodemographic variables or IQ. However, SDI performed below the controls in DF, DB, and FAB. The SDI were cognitively impaired in 3 of the 6 cognitive domains assessed by the FAB: abstract reasoning, motor programming, and cognitive flexibility. The FAB correlated with DF, SCWT, and WCST. In addition, some neuropsychological measures were correlated with the amount of alcohol, cannabis, and cocaine use. In conclusion, SDI performed more poorly than the comparison group on the FAB and the FAB's results were associated with other ECF-related tasks. The results suggested a negative impact of alcohol, cannabis, and cocaine use on the ECF. The FAB may be useful in assisting professionals as an instrument to screen for ECF-related deficits in SDI.

  9. Inhibitors bound to Ca(2+)-free sarcoplasmic reticulum Ca(2+)-ATPase lock its transmembrane region but not necessarily its cytosolic region, revealing the flexibility of the loops connecting transmembrane and cytosolic domains.

    PubMed

    Montigny, Cédric; Picard, Martin; Lenoir, Guillaume; Gauron, Carole; Toyoshima, Chikashi; Champeil, Philippe

    2007-12-25

    Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in the presence of inhibitors such as thapsigargin (TG), bound to the transmembrane region of this protein. Here, we examined the consequences of such binding for the protein. We found that, after TG binding, an active site ligand such as beryllium fluoride can still bind to the ATPase and change the conformation or dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but it becomes unable to induce any change in the transmembrane domain (as revealed by the intrinsic fluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changes normally induced by binding of MgATP or metal-free ATP, as well as those induced by binding of Mg2+ alone. In the nucleotide binding domain, the environment of Lys515 (as revealed by fluorescein isothiocyanate fluorescence after specific labeling of this residue) is significantly different in the ATPase complex with aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modified by TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolic domains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage by proteinase K at Thr242-Glu243, suggesting TG-induced reduction in the mobility of these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, inhibitors which also bind in or near the transmembrane region, also produce similar overall effects on Ca2+-free ATPase.

  10. Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility.

    PubMed

    Schneider, W P; Wensel, T G; Stryer, L; Oi, V T

    1988-04-01

    We have carried out nanosecond fluorescence polarization studies of genetically engineered immunoglobulins to determine the structural features controlling their segmental flexibility. The proteins studied were hybrids of a relatively rigid isotype (mouse IgG1) and a relatively flexible one (mouse IgG2a). They have identical light chains and heavy chain variable regions and have the same combining sites for epsilon-dansyl-L-lysine, a fluorescent hapten. The fluorescence of the bound dansyl chromophore was excited at 348 nm with subnanosecond laser pulses, and the emission in the nanosecond time range was measured with a single-photon-counting apparatus. The emission anisotropy kinetics of the hybrid antibodies revealed that segmental flexibility is controlled by the heavy chain constant region 1 (CH1) as well as by the hinge. In contrast, the CH2 and CH3 domains did not influence segmental flexibility. The hinge and CH1 domains must be properly matched to allow facile movement of the Fab units. Studies of hybrids of IgG1 and IgG2a within CH1 showed that the loop formed by residues 131-139 is important in controlling segmental flexibility. X-ray crystallographic studies by others of human IgG1 have shown that this loop makes several van der Waals contacts with the hinge.

  11. Joining RDC data from flexible protein domains

    NASA Astrophysics Data System (ADS)

    Sgheri, Luca

    2010-11-01

    We study the inverse problem of determining the conformational freedom of two protein domains from residual dipolar coupling (RDC) measurements. For each paramagnetic ion attached to one of the domains we obtain a magnetic susceptibility tensor χ from the RDC of couples of atoms of that domain, and a mean paramagnetic susceptibility tensor {\\bar{\\chi }} from the RDC of couples of atoms of the other domain. The latter is an integral average of rotations of χ which depends on the conformational freedom of the two domains. In this paper we consider the case when we have data from paramagnetic ions attached separately to each of the domains. We prove that in this case not all the elements of χ and {\\bar{\\chi }} are independent. We derive the mathematical equations for the compatibility of the measurements and show how these relations can be used in the presence of noisy data to determine a compatible set of χ and {\\bar{\\chi }} with an unconstrained minimization. If available, information about the shape of the noise can be included in the target function. We show that in this case the compatible set obtained has a reduced error with respect to the noisy data.

  12. Fuzzy domains: new way of describing flexibility and interdependence of the protein domains.

    PubMed

    Yesylevskyy, Semen O; Kharkyanen, Valery N

    2009-03-01

    We proposed the innovative method of domain identification based on the concept of the fuzzy domains. In this method each residue of the protein can belong to several domains simultaneously with certain weights, which reflect to what extent this residue shares the motion pattern of the given domain. Our method allows describing the fuzzy boundaries between the domains and the gradual changes of the motion pattern from one domain to the other. It provides the reasonable compromise between the continuous change of the protein dynamics from one residue to the other and the discrete description of the structure in terms of small number of domains. We suggested quantitative criterion, which shows the overall degree of domain flexibility in the protein. The concept of the fuzzy domains provides an innovative way of visualization of domain flexibility, which makes the gradual transitions between the domains clearly visible and comparable to available experimental and structural data. In the future, the concept of the fuzzy domains can be used in the coarse-grained simulations of the domain dynamics in order to account for internal protein flexibility.

  13. Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

    PubMed Central

    Ozawa, Kiyoshi; Horan, Nicholas P.; Robinson, Andrew; Yagi, Hiromasa; Hill, Flynn R.; Jergic, Slobodan; Xu, Zhi-Qiang; Loscha, Karin V.; Li, Nan; Tehei, Moeava; Oakley, Aaron J.; Otting, Gottfried; Huber, Thomas; Dixon, Nicholas E.

    2013-01-01

    A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data. PMID:23580545

  14. Binding to retinoblastoma pocket domain does not alter the inter-domain flexibility of the J domain of SV40 large T antigen.

    PubMed

    Williams, Christina K; Vaithiyalingam, Sivaraja; Hammel, Michal; Pipas, James; Chazin, Walter J

    2012-02-15

    Simian Virus 40 uses the large T antigen (Tag) to bind and inactivate retinoblastoma tumor suppressor proteins (Rb), which can result in cellular transformation. Tag is a modular protein with four domains connected by flexible linkers. The N-terminal J domain of Tag is necessary for Rb inactivation. Binding of Rb is mediated by an LXCXE consensus motif immediately C-terminal to the J domain. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) were used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N(260)) extending from the J domain through the origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the domains in N(260). Binding of pRb to a construct containing the LXCXE motif and the J domain revealed weak interactions between pRb and the J domain. Analysis of the complex of pRb and N(260) indicated that the OBD is not involved and retains its dynamic independence from the remainder of Tag. These results support a 'chaperone' model in which the J domain of Tag changes its orientation as it acts upon different protein complexes.

  15. Structure of a double-domain phosphagen kinase reveals an asymmetric arrangement of the tandem domains.

    PubMed

    Wang, Zhiming; Qiao, Zhu; Ye, Sheng; Zhang, Rongguang

    2015-04-01

    Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long α-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.

  16. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    PubMed Central

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  17. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain.

    PubMed

    Hilbert, Brendan J; Hayes, Janelle A; Stone, Nicholas P; Xu, Rui-Gang; Kelch, Brian A

    2017-01-12

    Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

  18. Architecture of cognitive flexibility revealed by lesion mapping.

    PubMed

    Barbey, Aron K; Colom, Roberto; Grafman, Jordan

    2013-11-15

    Neuroscience has made remarkable progress in understanding the architecture of human intelligence, identifying a distributed network of brain structures that support goal-directed, intelligent behavior. However, the neural foundations of cognitive flexibility and adaptive aspects of intellectual function remain to be well characterized. Here, we report a human lesion study (n=149) that investigates the neural bases of key competencies of cognitive flexibility (i.e., mental flexibility and the fluent generation of new ideas) and systematically examine their contributions to a broad spectrum of cognitive and social processes, including psychometric intelligence (Wechsler Adult Intelligence Scale), emotional intelligence (Mayer, Salovey, Caruso Emotional Intelligence Test), and personality (Neuroticism-Extraversion-Openness Personality Inventory). Latent variable modeling was applied to obtain error-free indices of each factor, followed by voxel-based lesion-symptom mapping to elucidate their neural substrates. Regression analyses revealed that latent scores for psychometric intelligence reliably predict latent scores for cognitive flexibility (adjusted R(2)=0.94). Lesion mapping results further indicated that these convergent processes depend on a shared network of frontal, temporal, and parietal regions, including white matter association tracts, which bind these areas into an integrated system. A targeted analysis of the unique variance explained by cognitive flexibility further revealed selective damage within the right superior temporal gyrus, a region known to support insight and the recognition of novel semantic relations. The observed findings motivate an integrative framework for understanding the neural foundations of adaptive behavior, suggesting that core elements of cognitive flexibility emerge from a distributed network of brain regions that support specific competencies for human intelligence.

  19. Architecture of cognitive flexibility revealed by lesion mapping

    PubMed Central

    Barbey, Aron K.; Colom, Roberto; Grafman, Jordan

    2013-01-01

    Neuroscience has made remarkable progress in understanding the architecture of human intelligence, identifying a distributed network of brain structures that support goal-directed, intelligent behavior. However, the neural foundations of cognitive flexibility and adaptive aspects of intellectual function remain to be well characterized. Here, we report a human lesion study (n = 149) that investigates the neural bases of key competencies of cognitive flexibility (i.e., mental flexibility and the fluent generation of new ideas) and systematically examine their contributions to a broad spectrum of cognitive and social processes, including psychometric intelligence (Wechsler Adult Intelligence Scale), emotional intelligence (Mayer, Salovey, Caruso Emotional Intelligence Test), and personality (Neuroticism–Extraversion–Openness Personality Inventory). Latent variable modeling was applied to obtain error-free indices of each factor, followed by voxel-based lesion-symptom mapping to elucidate their neural substrates. Regression analyses revealed that latent scores for psychometric intelligence reliably predict latent scores for cognitive flexibility (adjusted R2 = 0.94). Lesion mapping results further indicated that these convergent processes depend on a shared network of frontal, temporal, and parietal regions, including white matter association tracts, which bind these areas into an integrated system. A targeted analysis of the unique variance explained by cognitive flexibility further revealed selective damage within the right superior temporal gyrus, a region known to support insight and the recognition of novel semantic relations. The observed findings motivate an integrative framework for understanding the neural foundations of adaptive behavior, suggesting that core elements of cognitive flexibility emerge from a distributed network of brain regions that support specific competencies for human intelligence. PMID:23721727

  20. Frequency domain identification experiment on a large flexible structure

    NASA Technical Reports Server (NTRS)

    Bayard, D. S.; Hadaegh, F. Y.; Yam, Y.; Scheid, R. E.; Mettler, E.; Milman, M. H.

    1989-01-01

    Recent experiences in the field of flexible structure control in space have indicated a need for on-orbit system identification to support robust control redesign to avoid in-flight instabilities and maintain high spacecraft performance. The authors highlight an automated frequency domain system identification methodology recently developed to fill this need. The methodology supports (1) the estimation of system quantities useful for robust control analysis and design, (2) experiment design tailored to performing system identification in a typically constrained on-orbit environment, and (3) the automation of operations to reduce human-in-the-loop requirements. A basic overview of the methodology is presented first, followed by an experimental verification of the approach performed on the JPL/AFAL testbed facility.

  1. Crystal structure of Deinococcus radiodurans RecQ helicase catalytic core domain: the interdomain flexibility.

    PubMed

    Chen, Sheng-Chia; Huang, Chi-Hung; Yang, Chia Shin; Way, Tzong-Der; Chang, Ming-Chung; Chen, Yeh

    2014-01-01

    RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ from Deinococcus radiodurans (DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of the E. coli RecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

  2. Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

    PubMed

    Pessler, Frank; Hernandez, Nouria

    2003-08-01

    POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

  3. Probing protein flexibility reveals a mechanism for selective promiscuity

    PubMed Central

    Pabon, Nicolas A; Camacho, Carlos J

    2017-01-01

    Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

  4. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule.

    PubMed

    Jakob, Clarissa G; Edalji, Rohinton; Judge, Russell A; DiGiammarino, Enrico; Li, Yingchun; Gu, Jijie; Ghayur, Tariq

    2013-01-01

    Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.

  5. Structures of mesophilic and extremophilic citrate synthases reveal rigidity and flexibility for function.

    PubMed

    Wells, Stephen A; Crennell, Susan J; Danson, Michael J

    2014-10-01

    Citrate synthase (CS) catalyses the entry of carbon into the citric acid cycle and is highly-conserved structurally across the tree of life. Crystal structures of dimeric CSs are known in both "open" and "closed" forms, which differ by a substantial domain motion that closes the substrate-binding clefts. We explore both the static rigidity and the dynamic flexibility of CS structures from mesophilic and extremophilic organisms from all three evolutionary domains. The computational expense of this wide-ranging exploration is kept to a minimum by the use of rigidity analysis and rapid all-atom simulations of flexible motion, combining geometric simulation and elastic network modeling. CS structures from thermophiles display increased structural rigidity compared with the mesophilic enzyme. A CS structure from a psychrophile, stabilized by strong ionic interactions, appears to display likewise increased rigidity in conventional rigidity analysis; however, a novel modified analysis, taking into account the weakening of the hydrophobic effect at low temperatures, shows a more appropriate decreased rigidity. These rigidity variations do not, however, affect the character of the flexible dynamics, which are well conserved across all the structures studied. Simulation trajectories not only duplicate the crystallographically observed symmetric open-to-closed transitions, but also identify motions describing a previously unidentified antisymmetric functional motion. This antisymmetric motion would not be directly observed in crystallography but is revealed as an intrinsic property of the CS structure by modeling of flexible motion. This suggests that the functional motion closing the binding clefts in CS may be independent rather than symmetric and cooperative.

  6. Structure and Mutagenesis of Neural Cell Adhesion Molecule Domains Evidence for Flexibility in the Placement of Polysialic Acid Attachment Sites

    SciTech Connect

    Foley, Deirdre A.; Swartzentruber, Kristin G.; Lavie, Arnon; Colley, Karen J.

    2010-11-09

    The addition of {alpha}2,8-polysialic acid to the N-glycans of the neural cell adhesion molecule, NCAM, is critical for brain development and plays roles in synaptic plasticity, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our previous work indicates that the polysialylation of two N-glycans located on the fifth immunoglobulin domain (Ig5) of NCAM requires the presence of specific sequences in the adjacent fibronectin type III repeat (FN1). To understand the relationship of these two domains, we have solved the crystal structure of the NCAM Ig5-FN1 tandem. Unexpectedly, the structure reveals that the sites of Ig5 polysialylation are on the opposite face from the FN1 residues previously found to be critical for N-glycan polysialylation, suggesting that the Ig5-FN1 domain relationship may be flexible and/or that there is flexibility in the placement of Ig5 glycosylation sites for polysialylation. To test the latter possibility, new Ig5 glycosylation sites were engineered and their polysialylation tested. We observed some flexibility in glycosylation site location for polysialylation and demonstrate that the lack of polysialylation of a glycan attached to Asn-423 may be in part related to a lack of terminal processing. The data also suggest that, although the polysialyltransferases do not require the Ig5 domain for NCAM recognition, their ability to engage with this domain is necessary for polysialylation to occur on Ig5 N-glycans.

  7. Supermolecular bent configuration composed of achiral flexible liquid crystal trimers exhibiting chiral domains with opposite handedness.

    PubMed

    Sasaki, Haruna; Takanishi, Yoichi; Yamamoto, Jun; Yoshizawa, Atsushi

    2015-03-26

    Chirality's effects on physical properties of materials and how chirality arises have persisted as attractive issues in chemistry. We prepared a homologous series of achiral liquid crystal trimers in which three phenylpyrimidine units are connected via flexible heptamethylene spacers. An equimolecular mixture of a trimer with a nematic (N) phase and that with smectic A (SmA), smectic C (SmC), and smectic B phases was found to exhibit an N phase, a SmC phase, and a B4 phase composed of chiral domains with opposite handedness. The chiral characteristics of the B4 phase were confirmed by uncrossing the polarizers in opposite directions. XRD measurements reveal that both SmC and B4 phases have an interdigitated layer structure. That molecular interdigitation might form a supermolecular bent configuration that can produce saddle splay curvature to drive the B4 phase.

  8. Apo raver1 structure reveals distinct RRM domain orientations

    SciTech Connect

    Rangarajan, Erumbi S.; Lee, Jun Hyuck; Izard, Tina

    2012-09-17

    Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and {alpha}-actinin. The amino-terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1-3 domains in complex with Vt at 2.75 {angstrom} resolution. Here, we report crystal structure of the unbound raver1 RRM1-3 domains at 2 {angstrom} resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30{sup o}. Superposition with other RNA-bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three-tandem RRM domain structure.

  9. Discrimination reversal learning reveals greater female behavioural flexibility in guppies

    PubMed Central

    Lucon-Xiccato, Tyrone; Bisazza, Angelo

    2014-01-01

    Behavioural flexibility allows an animal to adapt its behaviour in response to changes in the environment. Research conducted in primates, rodents and domestic fowl suggests greater behavioural persistence and reduced behavioural flexibility in males. We investigated sex differences in behavioural flexibility in fish by comparing male and female guppies (Poecilia reticulata) in a reversal learning task. Fish were first trained on a colour discrimination, which was learned equally rapidly by males and females. However, once the reward contingency was reversed, females were better at inhibiting the previous response and reached criterion twice as fast as males. When reward reversing was repeated, males gradually reduced the number of errors, and the two sexes had a comparable performance after four reversals. We suggest that sex differences in behavioural flexibility in guppies can be explained in terms of the different roles that males and females play in reproduction.

  10. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  11. Eye Movements Reveal Components of Flexible Reading Strategies.

    ERIC Educational Resources Information Center

    Shebilske, Wayne L.; Fisher, Dennis F.

    The eye movements of two college graduates were monitored in a study of flexible reading, which is defined as the ability to adjust one's rate and approach to reading according to the purpose of reading, the difficulty of the material, and one's knowledge of the subject matter. The subjects were told to read an excerpt from a tenth grade biology…

  12. Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS13

    PubMed Central

    Deforche, L.; Roose, E.; Vandenbulcke, A.; Vandeputte, N.; Feys, H.B.; Springer, T.A.; Mi, L.Z.; Muia, J.; Sadler, J.E.; Soejima, K.; Rottensteiner, H.; Deckmyn, H.; De Meyer, S.F.; Vanhoorelbeke, K.

    2016-01-01

    Background Recently, conformational activation of ADAMTS13 was identified. This mechanism showed the evolution from a condensed and inhibited conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated structure due to ding with the von Willebrand factor (VWF). Objectives Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS13. Methods The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined in ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through RADAR analysis, tested in ELISA using deletion variants and visualized using electron microscopy. Results Eleven activating anti-ADAMTS13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains by electron microscopy furthermore supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, as activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. Conclusion Conformational activation of ADAMTS13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains. PMID:26391536

  13. Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates*

    PubMed Central

    Valnickova, Zuzana; Sanglas, Laura; Arolas, Joan L.; Petersen, Steen V.; Schar, Christine; Otzen, Daniel; Aviles, Francesc X.; Gomis-Rüth, F. Xavier; Enghild, Jan J.

    2010-01-01

    We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo. PMID:20880845

  14. Knowledge-Guided Docking of WW Domain Proteins and Flexible Ligands

    NASA Astrophysics Data System (ADS)

    Lu, Haiyun; Li, Hao; Banu Bte Sm Rashid, Shamima; Leow, Wee Kheng; Liou, Yih-Cherng

    Studies of interactions between protein domains and ligands are important in many aspects such as cellular signaling. We present a knowledge-guided approach for docking protein domains and flexible ligands. The approach is applied to the WW domain, a small protein module mediating signaling complexes which have been implicated in diseases such as muscular dystrophy and Liddle’s syndrome. The first stage of the approach employs a substring search for two binding grooves of WW domains and possible binding motifs of peptide ligands based on known features. The second stage aligns the ligand’s peptide backbone to the two binding grooves using a quasi-Newton constrained optimization algorithm. The backbone-aligned ligands produced serve as good starting points to the third stage which uses any flexible docking algorithm to perform the docking. The experimental results demonstrate that the backbone alignment method in the second stage performs better than conventional rigid superposition given two binding constraints. It is also shown that using the backbone-aligned ligands as initial configurations improves the flexible docking in the third stage. The presented approach can also be applied to other protein domains that involve binding of flexible ligand to two or more binding sites.

  15. Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis.

    PubMed

    Gahloth, Deepankar; Dunstan, Mark S; Quaglia, Daniela; Klumbys, Evaldas; Lockhart-Cairns, Michael P; Hill, Andrew M; Derrington, Sasha R; Scrutton, Nigel S; Turner, Nicholas J; Leys, David

    2017-09-01

    Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

  16. Controller design for flexible, distributed parameter mechanical arms via combined state space and frequency domain techniques

    NASA Technical Reports Server (NTRS)

    Book, W. J.; Majett, M.

    1982-01-01

    The potential benefits of the ability to control more flexible mechanical arms are discussed. A justification is made in terms of speed of movement. A new controller design procedure is then developed to provide this capability. It uses both a frequency domain representation and a state variable representation of the arm model. The frequency domain model is used to update the modal state variable model to insure decoupled states. The technique is applied to a simple example with encouraging results.

  17. Cognitive Flexibility and Undergraduate Physiology Students: Increasing Advanced Knowledge Acquisition within an Ill-Structured Domain

    ERIC Educational Resources Information Center

    Rhodes, Ashley E.; Rozell, Timothy G.

    2017-01-01

    Cognitive flexibility is defined as the ability to assimilate previously learned information and concepts to generate novel solutions to new problems. This skill is crucial for success within ill-structured domains such as biology, physiology, and medicine, where many concepts are simultaneously required for understanding a complex problem, yet…

  18. Tethered domains and flexible regions in tRNase Z(L), the long form of tRNase Z.

    PubMed

    Wilson, Christopher; Ramai, Daryl; Serjanov, Dmitri; Lama, Neema; Levinger, Louis; Chang, Emmanuel J

    2013-01-01

    tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3' trailer in a step central to tRNA maturation. The short form (tRNase Z(S)) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z(L)), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z(L) arose from a tandem duplication of tRNase Z(S) followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z(S) reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z(L) structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z(L) performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z(L) function.

  19. Tethered Domains and Flexible Regions in tRNase ZL, the Long Form of tRNase Z

    PubMed Central

    Wilson, Christopher; Ramai, Daryl; Serjanov, Dmitri; Lama, Neema; Levinger, Louis; Chang, Emmanuel J.

    2013-01-01

    tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3′ trailer in a step central to tRNA maturation. The short form (tRNase ZS) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase ZL), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase ZL arose from a tandem duplication of tRNase ZS followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase ZS reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase ZL structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase ZL performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase ZL function. PMID:23874404

  20. Time-domain simulation of acoustic wave propagation and interaction with flexible structures using Chebyshev collocation method

    NASA Astrophysics Data System (ADS)

    Wang, Chunqi; Huang, Lixi

    2012-09-01

    A time-domain Chebyshev collocation (ChC) method is used to simulate acoustic wave propagation and its interaction with flexible structures in ducts. The numerical formulation is described using a two-dimensional duct noise control system, which consists of an expansion chamber and a tensioned membrane covering the side-branch cavity. Full coupling between the acoustic wave and the structural vibration of the tensioned membrane is considered in the modelling. A systematic method of solution is developed for the discretized differential equations over multiple physical domains. The time-domain ChC model is tested against analytical solutions under two conditions: one with an initial state of wave motion; the other with a time-dependent acoustic source. Comparisons with the finite-difference time-domain (FDTD) method are also made. Results show that the time-domain ChC method is highly accurate and computationally efficient for the time-dependent solution of duct acoustic problems. For illustrative purposes, the time-domain ChC method is applied to investigate the acoustic performance of three typical duct noise control devices: the expansion chamber, the quarter wavelength resonator and the drum silencer. The time-dependent simulation of the sound-structure interaction in the drum silencer reveals the delicate role of the membrane mass and tension in its sound reflection capability.

  1. Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

    PubMed Central

    Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

  2. Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2.

    PubMed

    Poncet-Montange, Guillaume; Zhan, Yanai; Bardenhagen, Jennifer P; Petrocchi, Alessia; Leo, Elisabetta; Shi, Xi; Lee, Gilbert R; Leonard, Paul G; Geck Do, Mary K; Cardozo, Mario G; Andersen, Jannik N; Palmer, Wylie S; Jones, Philip; Ladbury, John E

    2015-03-01

    Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.

  3. Flexibility.

    ERIC Educational Resources Information Center

    Humphrey, L. Dennis

    1981-01-01

    Flexibility is an important aspect of all sports and recreational activities. Flexibility can be developed and maintained by stretching exercises. Exercises designed to develop flexibility in ankle joints, knees, hips, and the lower back are presented. (JN)

  4. Flexibility.

    ERIC Educational Resources Information Center

    Humphrey, L. Dennis

    1981-01-01

    Flexibility is an important aspect of all sports and recreational activities. Flexibility can be developed and maintained by stretching exercises. Exercises designed to develop flexibility in ankle joints, knees, hips, and the lower back are presented. (JN)

  5. Conformational selection of protein kinase A revealed by flexible-ligand flexible-protein docking.

    PubMed

    Huang, Zunnan; Wong, Chung F

    2009-03-01

    Protein kinases have high structural plasticity: their structure can change significantly, depending on what ligands are bound to them. Rigid-protein docking methods are not capable of describing such effects. Here, we present a new flexible-ligand flexible-protein docking model in which the protein can adopt conformations between two extremes observed experimentally. The model utilized a molecular dynamics-based simulated annealing cycling protocol and a distance-dependent dielectric model to perform docking. By testing this model on docking four diverse ligands to protein kinase A, we found that the ligands were able to dock successfully to the protein with the proper conformations of the protein induced. By imposing relatively soft conformational restraints to the protein during docking, this model reduced computational costs yet permitted essential conformational changes that were essential for these inhibitors to dock properly to the protein. For example, without adequate movement of the glycine-rich loop, it was difficult for the ligands to move from the surface of the protein to the binding site. In addition, these simulations called for better ways to compare simulation results with experiment other than using the popular root-mean-square deviation between the structure of a ligand in a docking pose and that in experiment because the structure of the protein also changed. In this work, we also calculated the correlation coefficient between protein-ligand/protein-protein distances in the docking structure and those in the crystal structure to check how well a ligand docked into the binding site of the protein and whether the proper conformation of the protein was induced.

  6. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    SciTech Connect

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard

    2013-09-15

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

  7. Active control for vibration suppression in a flexible beam using a modal domain optical fiber sensor

    NASA Technical Reports Server (NTRS)

    Cox, D. E.; Lindner, D. K.

    1991-01-01

    An account is given of the use of a modal-domain (MD) fiber-optic sensor as an active control system component for vibration suppression, whose output is proportional to the integral of the axial strain along the optical fiber. When an MD sensor is attached to, or embedded in, a flexible structure, it senses the strain in the structure along its gage length. On the basis of the present integration of the sensor model into a flexible-structure model, it becomes possible to design a control system with a dynamic compensator which adds damping to the low-order modes of the flexible structure. This modeling procedure has been experimentally validated.

  8. A time domain inverse dynamic method for the end point tracking control of a flexible manipulator

    NASA Technical Reports Server (NTRS)

    Kwon, Dong-Soo; Book, Wayne J.

    1991-01-01

    The inverse dynamic equation of a flexible manipulator was solved in the time domain. By dividing the inverse system equation into the causal part and the anticausal part, we calculated the torque and the trajectories of all state variables for a given end point trajectory. The interpretation of this method in the frequency domain was explained in detail using the two-sided Laplace transform and the convolution integral. The open loop control of the inverse dynamic method shows an excellent result in simulation. For real applications, a practical control strategy is proposed by adding a feedback tracking control loop to the inverse dynamic feedforward control, and its good experimental performance is presented.

  9. Cognitive flexibility and undergraduate physiology students: increasing advanced knowledge acquisition within an ill-structured domain.

    PubMed

    Rhodes, Ashley E; Rozell, Timothy G

    2017-09-01

    Cognitive flexibility is defined as the ability to assimilate previously learned information and concepts to generate novel solutions to new problems. This skill is crucial for success within ill-structured domains such as biology, physiology, and medicine, where many concepts are simultaneously required for understanding a complex problem, yet the problem consists of patterns or combinations of concepts that are not consistently used or needed across all examples. To succeed within ill-structured domains, a student must possess a certain level of cognitive flexibility: rigid thought processes and prepackaged informational retrieval schemes relying on rote memorization will not suffice. In this study, we assessed the cognitive flexibility of undergraduate physiology students using a validated instrument entitled Student's Approaches to Learning (SAL). The SAL evaluates how deeply and in what way information is processed, as well as the investment of time and mental energy that a student is willing to expend by measuring constructs such as elaboration and memorization. Our results indicate that students who rely primarily on memorization when learning new information have a smaller knowledge base about physiological concepts, as measured by a prior knowledge assessment and unit exams. However, students who rely primarily on elaboration when learning new information have a more well-developed knowledge base about physiological concepts, which is displayed by higher scores on a prior knowledge assessment and increased performance on unit exams. Thus students with increased elaboration skills possibly possess a higher level of cognitive flexibility and are more likely to succeed within ill-structured domains. Copyright © 2017 the American Physiological Society.

  10. Lid domain plasticity and lipid flexibility modulate enzyme specificity in human monoacylglycerol lipase.

    PubMed

    Riccardi, Laura; Arencibia, Jose M; Bono, Luca; Armirotti, Andrea; Girotto, Stefania; De Vivo, Marco

    2017-01-12

    Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named 'lid domain', which is structurally similar to that found in several other lipases. However, it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments, we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular, we clarify the key role of Phe159 and Ile179, two conserved residues within the lid domain, in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity.

  11. Synthetic actin-binding domains reveal compositional constraints for function.

    PubMed

    Lorenzi, Maria; Gimona, Mario

    2008-01-01

    The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.

  12. The Structure of the PapD-PapGII Pilin Complex Reveals an Open and Flexible P5 Pocket

    PubMed Central

    Ford, Bradley; Verger, Denis; Dodson, Karen; Volkan, Ender; Kostakioti, Maria; Elam, Jennifer; Pinkner, Jerome; Waksman, Gabriel

    2012-01-01

    P pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain's fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD's donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in–zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism. PMID:23002225

  13. Plant SAM-Domain Proteins Start to Reveal Their Roles.

    PubMed

    Denay, Grégoire; Vachon, Gilles; Dumas, Renaud; Zubieta, Chloe; Parcy, François

    2017-08-01

    Proteins often act in complexes assembled via protein-protein interaction domains. The sterile alpha motif (SAM) domain is one of the most prominent interaction domains in animals and is present in proteins of diverse functions. This domain allows head-to-tail closed oligomerisation or polymer formation resulting in homo- and/or heterocomplexes that have been shown to be important for proper protein localisation and function. In plants this domain is also present but has been poorly studied except for recent studies on the LEAFY floral regulator and the tRNA import component (TRIC)1/2 proteins. Here we catalogue SAM domain-containing proteins from arabidopsis (Arabidopsis thaliana), compare plant and other eukaryotic SAM domains, and perform homology modelling to probe plant SAM domain interaction capabilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Structural characterization of intramolecular Hg2+ transfer between flexibly-linked domains of mercuric ion reductase

    SciTech Connect

    Johs, Alexander; Harwood, Ian M; Parks, Jerry M; Nauss, Rachel; Smith, Jeremy C; Liang, Liyuan; Miller, Susan M

    2011-01-01

    The enzyme mercuric ion reductase, MerA, is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess a metallochaperone-like N-terminal domain, NmerA, that can transfer Hg2+ to the catalytic core (Core) for reduction to Hg0. These domains are tethered to the homodimeric Core by ~30-residue linkers that are subject to proteolysis, which has limited structural and functional characterization of the interactions of these domains. Here, we report purification of homogeneous full-length MerA using a fusion protein construct and combine small-angle X-ray and neutron scattering with molecular dynamics simulation to characterize the structure of constructs that mimic the system before and during handoff of Hg2+ from NmerA to the Core. The radii of gyration, distance distribution functions and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations resulting from flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg2+ from diverse protein substrates. Using a specific mutant of MerA allowed determination of the position and relative orientation of NmerA to the Core during Hg2+ handoff. The small buried surface area at the site of interaction suggests molecular recognition may be of less importance for the integrity of metal ion transfers between tethered domains than for transfers between separate proteins in metal trafficking pathways.

  15. Input preshaping with frequency domain information for flexible-link manipulator control

    NASA Technical Reports Server (NTRS)

    Tzes, Anthony; Englehart, Matthew J.; Yurkovich, Stephen

    1989-01-01

    The application of an input preshaping scheme to flexible manipulators is considered. The resulting control corresponds to a feedforward term that convolves in real-time the desired reference input with a sequence of impulses and produces a vibration free output. The robustness of the algorithm with respect to injected disturbances and modal frequency variations is not satisfactory and can be improved by convolving the input with a longer sequence of impulses. The incorporation of the preshaping scheme to a closed-loop plant, using acceleration feedback, offers satisfactory disturbance rejection due to feedback and cancellation of the flexible mode effects due to the preshaping. A frequency domain identification scheme is used to estimate the modal frequencies on-line and subsequently update the spacing between the impulses. The combined adaptive input preshaping scheme provides the fastest possible slew that results in a vibration free output.

  16. Controlling conformational flexibility of an O₂-binding H-NOX domain.

    PubMed

    Weinert, Emily E; Phillips-Piro, Christine M; Tran, Rosalie; Mathies, Richard A; Marletta, Michael A

    2011-08-16

    Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.

  17. Frequency domain active vibration control of a flexible plate based on neural networks

    NASA Astrophysics Data System (ADS)

    Liu, Jinxin; Chen, Xuefeng; He, Zhengjia

    2013-06-01

    A neural-network (NN)-based active control system was proposed to reduce the low frequency noise radiation of the simply supported flexible plate. Feedback control system was built, in which neural network controller (NNC) and neural network identifier (NNI) were applied. Multi-frequency control in frequency domain was achieved by simulation through the NN-based control systems. A pre-testing experiment of the control system on a real simply supported plate was conducted. The NN-based control algorithm was shown to perform effectively. These works lay a solid foundation for the active vibration control of mechanical structures.

  18. NMR spectroscopic and bioinformatic analyses of the LTBP1 C-terminus reveal a highly dynamic domain organisation.

    PubMed

    Robertson, Ian B; Handford, Penny A; Redfield, Christina

    2014-01-01

    Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. (15)N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible "knotted rope" structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.

  19. Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences.

    PubMed

    Choudhury, Susobhan; Ghosh, Basusree; Singh, Priya; Ghosh, Raka; Roy, Siddhartha; Pal, Samir Kumar

    2016-07-21

    The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA.

  20. Novel domain formation reveals proto-architecture in inferotemporal cortex

    PubMed Central

    Srihasam, Krishna; Vincent, Justin L.; Livingstone, Margaret S.

    2014-01-01

    Primate inferotemporal cortex is subdivided into domains for biologically important categories, like faces, bodies, and scenes, as well as domains for culturally entrained categories, like text or buildings. These domains are in stereotyped locations in most humans and monkeys. To ask what determines the location of such domains, we intensively trained 7 juvenile monkeys to recognize 3 distinct sets of shapes. After training, the monkeys developed regions that were selectively responsive to each trained set. The location of each specialization was similar across monkeys, despite differences in training order. This indicates that the location of training effects does not depend on function or expertise, but rather some kind of proto-organization. We explore the possibility that this proto-organization is retinotopic or shape-based. PMID:25362472

  1. Nanoviscosity Measurements Revealing Domain Formation in Biomimetic Membranes.

    PubMed

    Hasan, Imad Younus; Mechler, Adam

    2017-02-07

    Partitioning of lipid molecules in biomimetic membranes is a model system for the study of naturally occurring domains, such as rafts, in biological membranes. The existence of nanometer scale membrane domains in binary lipid mixtures has been shown with microscopy methods; however, the nature of these domains has not been established unequivocally. A common notion is to ascribe domain separation to thermodynamic phase equilibria. However, characterizing thermodynamic phases of single bilayer membranes has not been possible due to their extreme dimensions: the size of the domains falls to the order of tens to hundreds of nanometers whereas the membrane thickness is only a few nanometers. Here, we present direct measurements of phase transitions in single bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid mixtures using quartz crystal microbalance-based nanoviscosity measurements. Coexisting thermodynamic phases have been successfully identified, and a phase diagram was constructed for the single bilayer binary lipid system. It was demonstrated that domain separation only takes place in planar membranes, and thus, it is absent in liposomes and not detectable in calorimetric measurements on liposome suspensions. On the basis of energetic analysis, the main transition was identified as the breaking of van der Waals interactions between the acyl chains.

  2. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  3. Determination of modal residues and residual flexibility for time-domain system realization

    SciTech Connect

    Alvin, K.F.; Peterson, L.D.

    1995-05-01

    A linear least-squares procedure for the determination of modal residues using time-domain system realization theory is presented. The present procedure is shown to be theoretically equivalent to residue determination in realization algorithms such as the Eigensystem Realization Algorithm (ERA) and Q-Markov COVER. However, isolating the optimal residue estimation problem from the general realization problem affords several advantages over standard realization algorithms for structural dynamics identification. Primary among these are the ability to identify data sets with large numbers of sensors using small numbers of reference point responses, and the inclusion of terms which accurately model the effects of residual flexibility. The accuracy and efficiency of the present realization theory-based procedure is demonstrated for both simulated and experimental data.

  4. Structural insights into the role of domain flexibility in human DNA ligase IV.

    PubMed

    Ochi, Takashi; Wu, Qian; Chirgadze, Dimitri Y; Grossmann, J Günter; Bolanos-Garcia, Victor M; Blundell, Tom L

    2012-07-03

    Knowledge of the architecture of DNA ligase IV (LigIV) and interactions with XRCC4 and XLF-Cernunnos is necessary for understanding its role in the ligation of double-strand breaks during nonhomologous end joining. Here we report the structure of a subdomain of the nucleotidyltrasferase domain of human LigIV and provide insights into the residues associated with LIG4 syndrome. We use this structural information together with the known structures of the BRCT/XRCC4 complex and those of LigIV orthologs to interpret small-angle X-ray scattering of LigIV in complex with XRCC4 and size exclusion chromatography of LigIV, XRCC4, and XLF-Cernunnos. Our results suggest that the flexibility of the catalytic region is limited in a manner that affects the formation of the LigIV/XRCC4/XLF-Cernunnos complex.

  5. Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

    PubMed Central

    Angus, Allan G. N.; Loquet, Antoine; Stack, Séamus J.; Dalrymple, David; Gatherer, Derek

    2012-01-01

    Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ∼2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helix-loop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity. PMID:22072760

  6. Localized force application reveals mechanically sensitive domains of Piezo1

    PubMed Central

    Wu, Jason; Goyal, Raman; Grandl, Jörg

    2016-01-01

    Piezos are mechanically activated ion channels that function as sensors of touch and pressure in various cell types. However, the precise mechanism and structures mediating mechanical activation and subsequent inactivation have not yet been identified. Here we use magnetic nanoparticles as localized transducers of mechanical force in combination with pressure-clamp electrophysiology to identify mechanically sensitive domains important for activation and inactivation. PMID:27694883

  7. WW domain folding complexity revealed by infrared spectroscopy.

    PubMed

    Davis, Caitlin M; Dyer, R Brian

    2014-09-02

    Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.

  8. WW Domain Folding Complexity Revealed by Infrared Spectroscopy

    PubMed Central

    2015-01-01

    Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics. PMID:25121968

  9. The utility of flexible sigmoidoscopy after a computerized tomographic colonography revealing only rectosigmoid lesions.

    PubMed

    Young, P E; Gentry, A B; Cash, B D

    2008-03-15

    Identifying polyps by computerized tomographic colonography typically prompts colonoscopy, increasing its cost, risk and inconvenience. Many polyps are confined to the rectosigmoid and theoretically amenable to resection via flexible sigmoidoscopy. To determine the prevalence of advanced proximal colonic neoplasia when computerized tomographic colonography reveals only rectosigmoid polyps, and characterize the yield of polypectomy via flexible sigmoidoscopy in such patients. Subjects underwent computerized tomographic colonography and colonoscopy with segmental unblinding. Patients with only rectosigmoid findings by computerized tomographic colonography were identified retrospectively. Flexible sigmoidoscopy findings were estimated by including lesions distal to the descending/sigmoid colon junction during colonoscopy. Proximal lesions were also reviewed. Advanced lesions were defined as: adenocarcinoma, tubular adenoma >1 cm, > or =3 tubular adenomas, tubulovillous histology or high-grade dysplasia. By computerized tomographic colonography, 15% (203 of 1372) had only rectosigmoid polyps. Concomitant lesions in the proximal colon were seen in 32% (64 of 203) during colonoscopy. Advanced proximal neoplasia occurred in 2% (three of 203) with only rectosigmoid polyps on computerized tomographic colonography. Using flexible sigmoidoscopy to follow-up computerized tomographic colonography demonstrating only rectosigmoid polyps would eliminate 15% of subsequent colonoscopies. This strategy carries a small risk of missed proximal advanced neoplasia. This miss rate appears comparable to that of colonoscopy alone. Further study on the cost-effectiveness of this approach is warranted.

  10. Binding of flexible and constrained ligands to the Grb2 SH2 domain: structural effects of ligand preorganization

    PubMed Central

    Clements, John H.; DeLorbe, John E.; Benfield, Aaron P.; Martin, Stephen F.

    2010-01-01

    Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH2 (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein–ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of each flexible/constrained ligand pair were generally similar to those observed upon comparing such contacts in coexisting complexes. The average adjusted B factors of the backbone atoms of the domain and loop regions are significantly greater in the complexes of constrained ligands than in the complexes of

  11. Evolutionary versatility of eukaryotic protein domains revealed by their bigram networks

    PubMed Central

    2011-01-01

    Background Protein domains are globular structures of independently folded polypeptides that exert catalytic or binding activities. Their sequences are recognized as evolutionary units that, through genome recombination, constitute protein repertoires of linkage patterns. Via mutations, domains acquire modified functions that contribute to the fitness of cells and organisms. Recent studies have addressed the evolutionary selection that may have shaped the functions of individual domains and the emergence of particular domain combinations, which led to new cellular functions in multi-cellular animals. This study focuses on modeling domain linkage globally and investigates evolutionary implications that may be revealed by novel computational analysis. Results A survey of 77 completely sequenced eukaryotic genomes implies a potential hierarchical and modular organization of biological functions in most living organisms. Domains in a genome or multiple genomes are modeled as a network of hetero-duplex covalent linkages, termed bigrams. A novel computational technique is introduced to decompose such networks, whereby the notion of domain "networking versatility" is derived and measured. The most and least "versatile" domains (termed "core domains" and "peripheral domains" respectively) are examined both computationally via sequence conservation measures and experimentally using selected domains. Our study suggests that such a versatility measure extracted from the bigram networks correlates with the adaptivity of domains during evolution, where the network core domains are highly adaptive, significantly contrasting the network peripheral domains. Conclusions Domain recombination has played a major part in the evolution of eukaryotes attributing to genome complexity. From a system point of view, as the results of selection and constant refinement, networks of domain linkage are structured in a hierarchical modular fashion. Domains with high degree of networking

  12. A selective screen reveals discrete functional domains in Drosophila Nanos.

    PubMed Central

    Arrizabalaga, G; Lehmann, R

    1999-01-01

    The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis. PMID:10581288

  13. Flexibility of the cytoplasmic domain of the phototaxis transducer II from Natronomonas pharaonis.

    PubMed

    Budyak, Ivan L; Mironova, Olga S; Yanamala, Naveena; Manoharan, Vijayalaxmi; Büldt, Georg; Schlesinger, Ramona; Klein-Seetharaman, Judith

    2008-01-01

    Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family.

  14. The Vanderbilt Expertise Test Reveals Domain-General and Domain-Specific Sex Effects in Object Recognition

    PubMed Central

    McGugin, Rankin W.; Richler, Jennifer J.; Herzmann, Grit; Speegle, Magen; Gauthier, Isabel

    2012-01-01

    Individual differences in face recognition are often contrasted with differences in object recognition using a single object category. Likewise, individual differences in perceptual expertise for a given object domain have typically been measured relative to only a single category baseline. In Experiment 1, we present a new test of object recognition, the Vanderbilt Expertise Test (VET), which is comparable in methods to the Cambridge Face Memory Task (CFMT) but uses eight different object categories. Principal component analysis reveals that the underlying structure of the VET can be largely explained by two independent factors, which demonstrate good reliability and capture interesting sex differences inherent in the VET structure. In Experiment 2, we show how the VET can be used to separate domain-specific from domain-general contributions to a standard measure of perceptual expertise. While domain-specific contributions are found for car matching for both men and women and for plane matching in men, women in this sample appear to use more domain-general strategies to match planes. In Experiment 3, we use the VET to demonstrate that holistic processing of faces predicts face recognition independently of general object recognition ability, which has a sex-specific contribution to face recognition. Overall, the results suggest that the VET is a reliable and valid measure of object recognition abilities and can measure both domain-general skills and domain-specific expertise, which were both found to depend on the sex of observers. PMID:22877929

  15. The Vanderbilt Expertise Test reveals domain-general and domain-specific sex effects in object recognition.

    PubMed

    McGugin, Rankin W; Richler, Jennifer J; Herzmann, Grit; Speegle, Magen; Gauthier, Isabel

    2012-09-15

    Individual differences in face recognition are often contrasted with differences in object recognition using a single object category. Likewise, individual differences in perceptual expertise for a given object domain have typically been measured relative to only a single category baseline. In Experiment 1, we present a new test of object recognition, the Vanderbilt Expertise Test (VET), which is comparable in methods to the Cambridge Face Memory Task (CFMT) but uses eight different object categories. Principal component analysis reveals that the underlying structure of the VET can be largely explained by two independent factors, which demonstrate good reliability and capture interesting sex differences inherent in the VET structure. In Experiment 2, we show how the VET can be used to separate domain-specific from domain-general contributions to a standard measure of perceptual expertise. While domain-specific contributions are found for car matching for both men and women and for plane matching in men, women in this sample appear to use more domain-general strategies to match planes. In Experiment 3, we use the VET to demonstrate that holistic processing of faces predicts face recognition independently of general object recognition ability, which has a sex-specific contribution to face recognition. Overall, the results suggest that the VET is a reliable and valid measure of object recognition abilities and can measure both domain-general skills and domain-specific expertise, which were both found to depend on the sex of observers.

  16. How Conformational Flexibility Stabilizes the Hyperthermophilic Elongation Factor G-domain

    PubMed Central

    Kalimeri, Maria; Rahaman, Obaidur; Melchionna, Simone; Sterpone, Fabio

    2014-01-01

    Proteins from thermophilic organisms are stable and functional well above ambient temperature. Understanding the molecular mechanism underlying such a resistance is of crucial interest for many technological applications. For some time, thermal stability has been assumed to correlate with high mechanical rigidity of the protein matrix. In this work we address this common belief by carefully studying a pair of homologous G-domain proteins, with their melting temperatures differing by 40 K. To probe the thermal-stability content of the two proteins we use extensive simulations covering the microsecond time range and employ several different indicators to assess the salient features of the conformational landscape and the role of internal fluctuations at ambient condition. At the atomistic level, while the magnitude of fluctuations is comparable, the distribution of flexible and rigid stretches of amino-acids is more regular in the thermophilic protein causing a cage-like correlation of amplitudes along the sequence. This caging effect is suggested to favor stability at high T by confining the mechanical excitations. Moreover, it is found that the thermophilic protein, when folded, visits a higher number of conformational substates than the mesophilic homologue. The entropy associated with the occupation of the different substates, along with the thermal resilience of the protein intrinsic compressibility, provide a qualitative insight on the thermal stability of the thermophilic protein as compared to its mesophilic homologue. Our findings potentially open the route to new strategies in the design of thermostable proteins. PMID:24087838

  17. Solution NMR of MPS-1 reveals a random coil cytosolic domain structure.

    PubMed

    Li, Pan; Shi, Pan; Lai, Chaohua; Li, Juan; Zheng, Yuanyuan; Xiong, Ying; Zhang, Longhua; Tian, Changlin

    2014-01-01

    Caenorhabditis elegans MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member) auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134-256) in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity.

  18. Solution NMR of MPS-1 Reveals a Random Coil Cytosolic Domain Structure

    PubMed Central

    Lai, Chaohua; Li, Juan; Zheng, Yuanyuan; Xiong, Ying; Zhang, Longhua; Tian, Changlin

    2014-01-01

    Caenorhabditis elegans MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member) auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134–256) in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity. PMID:25347290

  19. Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies.

    PubMed

    Sigman, Jeffrey A; Patwa, Tasneem H; Tablante, Ana V; Joseph, Calleen D; Glucksman, Marc J; Wolfson, Adele J

    2005-05-15

    Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.

  20. Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.

    PubMed

    Scott, Daniel; Layfield, Robert; Oldham, Neil J

    2015-08-01

    Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states.

  1. Maximum occurrence analysis of protein conformations for different distributions of paramagnetic metal ions within flexible two-domain proteins.

    PubMed

    Luchinat, Claudio; Nagulapalli, Malini; Parigi, Giacomo; Sgheri, Luca

    2012-02-01

    Multidomain proteins are composed of rigid domains connected by (flexible) linkers. Therefore, the domains may experience a large degree of reciprocal reorientation. Pseudocontact shifts and residual dipolar couplings arising from one or more paramagnetic metals successively placed in a single metal binding site in the protein can be used as restraints to assess the degree of mobility of the different domains. They can be used to determine the maximum occurrence (MO) of each possible protein conformation, i.e. the maximum weight that such conformations can have independently of the real structural ensemble, in agreement with the provided restraints. In the case of two-domain proteins, the metal ions can be placed all in the same domain, or distributed between the two domains. It has been demonstrated that the quantity of independent information for the characterization of the system is larger when all metals are bound in the same domain. At the same time, it has been shown that there are practical advantages in placing the metals in different domains. Here, it is shown that distributing the metals between the domains provides a tool for defining a coefficient of compatibility among the restraints obtained from different metals, without a significant decrease of the capability of the MO values to discriminate among conformations with different weights.

  2. A domain-centric analysis of oomycete plant pathogen genomes reveals unique protein organization.

    PubMed

    Seidl, Michael F; Van den Ackerveken, Guido; Govers, Francine; Snel, Berend

    2011-02-01

    Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens.

  3. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant

    PubMed Central

    Yuchi, Zhiguang; Yuen, Siobhan M. Wong King; Lau, Kelvin; Underhill, Ainsley Q.; Cornea, Razvan L.; Fessenden, James D.; Van Petegem, Filip

    2015-01-01

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca2+-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2–1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding. PMID:26245150

  4. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant.

    PubMed

    Yuchi, Zhiguang; Yuen, Siobhan M Wong King; Lau, Kelvin; Underhill, Ainsley Q; Cornea, Razvan L; Fessenden, James D; Van Petegem, Filip

    2015-08-06

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca(2+)-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

  5. Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain1[OPEN

    PubMed Central

    Villarino, Gonzalo H.; Hu, Qiwen; Flores-Vergara, Miguel; Sehra, Bhupinder; Brumos, Javier; Stepanova, Anna N.; Sundberg, Eva; Heber, Steffen

    2016-01-01

    Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis. PMID:26983993

  6. Exploring Flexibility of Progesterone Receptor Ligand Binding Domain Using Molecular Dynamics

    PubMed Central

    Zheng, Liangzhen; Mu, Yuguang

    2016-01-01

    Progesterone receptor (PR), a member of nuclear receptor (NR) superfamily, plays a vital role for female reproductive tissue development, differentiation and maintenance. PR ligand, such as progesterone, induces conformation changes in PR ligand binding domain (LBD), thus mediates subsequent gene regulation cascades. PR LBD may adopt different conformations upon an agonist or an antagonist binding. These different conformations would trigger distinct transcription events. Therefore, the dynamics of PR LBD would be of general interest to biologists for a deep understanding of its structure-function relationship. However, no apo-form (non-ligand bound) of PR LBD model has been proposed either by experiments or computational methods so far. In this study, we explored the structural dynamics of PR LBD using molecular dynamics simulations and advanced sampling tools in both ligand-bound and the apo-forms. Resolved by the simulation study, helix 11, helix 12 and loop 895–908 (the loop between these two helices) are quite flexible in antagonistic conformation. Several residues, such as Arg899 and Glu723, could form salt-bridging interaction between helix 11 and helix 3, and are important for the PR LBD dynamics. And we also propose that helix 12 in apo-form PR LBD, not like other NR LBDs, such as human estrogen receptor α (ERα) LBD, may not adopt a totally extended conformation. With the aid of umbrella sampling and metadynamics simulations, several stable conformations of apo-form PR LBD have been sampled, which may work as critical structural models for further large scale virtual screening study to discover novel PR ligands for therapeutic application. PMID:27824891

  7. The Crystal Structures of EAP Domains from Staphylococcus aureus Reveal an Unexpected Homology to Bacterial Superantigens

    SciTech Connect

    Geisbrecht, B V; Hamaoka, B Y; Perman, B; Zemla, A; Leahy, D J

    2005-10-14

    The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 {angstrom} resolution, respectively. These structures reveal a core fold that is comprised of an {alpha}-helix lying diagonally across a five-stranded, mixed {beta}-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the {beta}-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

  8. Flexibility and non-destructive conductivity measurements of Ag nanowire based transparent conductive films via terahertz time domain spectroscopy.

    PubMed

    Hwang, Gyujeong; Balci, Soner; Güngördü, M Zeki; Maleski, Alex; Waters, Joseph; Lee, Sunjong; Choi, Sangjun; Kim, Kyoungkook; Cho, Soohaeng; Kim, Seongsin M

    2017-02-20

    Highly stable and flexible transparent electrodes are fabricated based on silver nanowires (AgNWs) on both polyethylene-terephthalate (PET) and polyimide (PI) substrates. Terahertz time domain spectroscopy (THz-TDS) was utilized to probe AgNW films while bended with a radius 5 mm to discover conductivity of bended films which was further analyzed through Drude-Smith model. AgNW films experience little degradation in conductivity (<3%) before, after, and during 1000 bending cycles. Highly stable AgNW flexible electrodes have broad applications in flexible optoelectronic and electronic devices. THz-TDS is an effective technique to investigate the electrical properties of the bended and flattened conducting films in a nondestructive manner.

  9. A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering.

    PubMed

    Borges, Júlio C; Seraphim, Thiago V; Dores-Silva, Paulo R; Barbosa, Leandro R S

    2016-06-01

    Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.

  10. Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

    DOE PAGES

    Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

    2015-01-22

    F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

  11. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    SciTech Connect

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H.

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  12. IQGAP proteins reveal an atypical phosphoinositide (aPI) binding domain with a pseudo C2 domain fold.

    PubMed

    Dixon, Miles J; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R; van Aalten, Daan M F; Downes, C Peter; Batty, Ian H

    2012-06-29

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  13. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold*

    PubMed Central

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M. F.; Downes, C. Peter; Batty, Ian H.

    2012-01-01

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105–107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

  14. Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks*

    PubMed Central

    Abu-Odeh, Mohammad; Bar-Mag, Tomer; Huang, Haiming; Kim, TaeHyung; Salah, Zaidoun; Abdeen, Suhaib K.; Sudol, Marius; Reichmann, Dana; Sidhu, Sachdev; Kim, Philip M.; Aqeilan, Rami I.

    2014-01-01

    WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. PMID:24550385

  15. Characterizing WW domain interactions of tumor suppressor WWOX reveals its association with multiprotein networks.

    PubMed

    Abu-Odeh, Mohammad; Bar-Mag, Tomer; Huang, Haiming; Kim, TaeHyung; Salah, Zaidoun; Abdeen, Suhaib K; Sudol, Marius; Reichmann, Dana; Sidhu, Sachdev; Kim, Philip M; Aqeilan, Rami I

    2014-03-28

    WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks.

  16. Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy

    PubMed Central

    Bu, Zimei; Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Callaway, David J. E.

    2005-01-01

    Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 Å to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques. PMID:16306270

  17. Solution structure of the Big domain from Streptococcus pneumoniae reveals a novel Ca2+-binding module

    PubMed Central

    Wang, Tao; Zhang, Jiahai; Zhang, Xuecheng; Xu, Chao; Tu, Xiaoming

    2013-01-01

    Streptococcus pneumoniae is a pathogen causing acute respiratory infection, otitis media and some other severe diseases in human. In this study, the solution structure of a bacterial immunoglobulin-like (Big) domain from a putative S. pneumoniae surface protein SP0498 was determined by NMR spectroscopy. SP0498 Big domain adopts an eight-β-strand barrel-like fold, which is different in some aspects from the two-sheet sandwich-like fold of the canonical Ig-like domains. Intriguingly, we identified that the SP0498 Big domain was a Ca2+ binding domain. The structure of the Big domain is different from those of the well known Ca2+ binding domains, therefore revealing a novel Ca2+-binding module. Furthermore, we identified the critical residues responsible for the binding to Ca2+. We are the first to report the interactions between the Big domain and Ca2+ in terms of structure, suggesting an important role of the Big domain in many essential calcium-dependent cellular processes such as pathogenesis. PMID:23326635

  18. Conserved tertiary couplings stabilize elements in the PDZ fold, leading to characteristic patterns of domain conformational flexibility.

    PubMed

    Ho, Bosco K; Agard, David A

    2010-03-01

    Single-domain allostery has been postulated to occur through intramolecular pathways of signaling within a protein structure. We had previously investigated these pathways by introducing a local thermal perturbation and analyzed the anisotropic propagation of structural changes throughout the protein. Here, we develop an improved approach, the Rotamerically Induced Perturbation (RIP), that identifies strong couplings between residues by analyzing the pathways of heat-flow resulting from thermal excitation of rotameric rotations at individual residues. To explore the nature of these couplings, we calculate the complete coupling maps of 5 different PDZ domains. Although the PDZ domain is a well conserved structural fold that serves as a scaffold in many protein-protein complexes, different PDZ domains display unique patterns of conformational flexibility in response to ligand binding: some show a significant shift in a set of alpha-helices, while others do not. Analysis of the coupling maps suggests a simple relationship between the computed couplings and observed conformational flexibility. In domains where the alpha-helices are rigid, we find couplings of the alpha-helices to the body of the protein, whereas in domains having ligand-responsive alpha-helices, no couplings are found. This leads to a model where the alpha-helices are intrinsically dynamic but can be damped if sidechains interact at key tertiary contacts. These tertiary contacts correlate to high covariation contacts as identified by the statistical coupling analysis method. As these dynamic modules are exploited by various allosteric mechanisms, these tertiary contacts have been conserved by evolution.

  19. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    SciTech Connect

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans

    2014-07-01

    Pyrin domains (PYDs) recruit downstream effector molecules in NLR signalling. A specific charge-relay system suggests a the formation of a signalling complex involving a PYD dimer. The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.

  20. Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis

    PubMed Central

    Díaz-Espinoza, Rodrigo; Garcés, Andrea P.; Arbildua, José J.; Montecinos, Felipe; Brunet, Juan E.; Lagos, Rosalba; Monasterio, Octavio

    2007-01-01

    FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions. PMID:17656575

  1. Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility.

    PubMed

    Paredes, Diana I; Watters, Kyle; Pitman, Derek J; Bystroff, Christopher; Dordick, Jonathan S

    2011-10-20

    Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes. We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes. These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

  2. Biomimetic and Live Medusae Reveal the Mechanistic Advantages of a Flexible Bell Margin

    PubMed Central

    Colin, Sean P.; Costello, John H.; Dabiri, John O.; Villanueva, Alex; Blottman, John B.; Gemmell, Brad J.; Priya, Shashank

    2012-01-01

    Flexible bell margins are characteristic components of rowing medusan morphologies and are expected to contribute towards their high propulsive efficiency. However, the mechanistic basis of thrust augmentation by flexible propulsors remained unresolved, so the impact of bell margin flexibility on medusan swimming has also remained unresolved. We used biomimetic robotic jellyfish vehicles to elucidate that propulsive thrust enhancement by flexible medusan bell margins relies upon fluid dynamic interactions between entrained flows at the inflexion point of the exumbrella and flows expelled from under the bell. Coalescence of flows from these two regions resulted in enhanced fluid circulation and, therefore, thrust augmentation for flexible margins of both medusan vehicles and living medusae. Using particle image velocimetry (PIV) data we estimated pressure fields to demonstrate a mechanistic basis of enhanced flows associated with the flexible bell margin. Performance of vehicles with flexible margins was further enhanced by vortex interactions that occur during bell expansion. Hydrodynamic and performance similarities between robotic vehicles and live animals demonstrated that the propulsive advantages of flexible margins found in nature can be emulated by human-engineered propulsors. Although medusae are simple animal models for description of this process, these results may contribute towards understanding the performance of flexible margins among other animal lineages. PMID:23145016

  3. Conformational Flexibility in the Flap Domains of Ligand-Free HIV Protease

    SciTech Connect

    Heaslet, H.; Rosenfeld, R.; Giffin, M.; Lin, Y.-C.; Tam, K.; Torbett, B.E.; Elder, J.H.; Stout, C.D.

    2009-06-01

    The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15 {angstrom} resolution are reported. In one crystal form HIV PR adopts an 'open' conformation with a 7.7 {angstrom} separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are 'curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3 {angstrom} resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. The mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a 'curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the 'open' and 'curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire {beta}-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the {beta}-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors.

  4. The crystal structure of Aspergillus fumigatus cyclophilin reveals 3D domain swapping of a central element.

    PubMed

    Limacher, Andreas; Kloer, Daniel P; Flückiger, Sabine; Folkers, Gerd; Crameri, Reto; Scapozza, Leonardo

    2006-02-01

    The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

  5. Replicate altitudinal clines reveal that evolutionary flexibility underlies adaptation to drought stress in annual Mimulus guttatus.

    PubMed

    Kooyers, Nicholas J; Greenlee, Anna B; Colicchio, Jack M; Oh, Morgan; Blackman, Benjamin K

    2015-04-01

    Examining how morphology, life history and physiology vary along environmental clines can reveal functional insight into adaptations to climate and thus inform predictions about evolutionary responses to global change. Widespread species occurring over latitudinal and altitudinal gradients in seasonal water availability are excellent systems for investigating multivariate adaptation to drought stress. Under common garden conditions, we characterized variation in 27 traits for 52 annual populations of Mimulus guttatus sampled from 10 altitudinal transects. We also assessed variation in the critical photoperiod for flowering and surveyed neutral genetic markers to control for demography when analyzing clinal patterns. Many drought escape (e.g. flowering time) and drought avoidance (e.g. specific leaf area, succulence) traits exhibited geographic or climatic clines, which often remained significant after accounting for population structure. Critical photoperiod and flowering time in glasshouse conditions followed distinct clinal patterns, indicating different aspects of seasonal phenology confer adaptation to unique agents of selection. Although escape and avoidance traits were negatively correlated range-wide, populations from sites with short growing seasons produced both early flowering and dehydration avoidance phenotypes. Our results highlight how abundant genetic variation in the component traits that build multivariate adaptations to drought stress provides flexibility for intraspecific adaptation to diverse climates. © 2014 The Authors New Phytologist © 2014 New Phytologist Trust.

  6. Torsional flexibility of B-DNA as revealed by conformational analysis.

    PubMed Central

    Zhurkin, V B; Lysov, Y P; Florentiev, V L; Ivanov, V I

    1982-01-01

    The thermal fluctuations of a regular double helix belonging to the B-family were studied by means of atom-atomic potentials method. The winding angle fluctuation was found to be 2.4 degrees for poly(dA):poly(dT) and 3.0 degrees for poly(dG):poly(dC). The reasonable agreement of these estimations with those obtained experimentally reveals the essential role of the small-amplitude torsional vibrations of atoms in the mechanism of the double helix flexibility. The calculated equilibrium winding angle, tau 0, essentially depends on the degree of neutralization of phosphate groups, being about 35.5 degrees for the full neutralization. The deoxyribose pucker is closely related to the tau angle: while tau proceeds from 30 degrees to 45 degrees the pseudorotation phase angle, P, increases from 126 degrees to 164 degrees. Fluctuations of the angles TL and TW, which specify inclination of the bases to the helix axis, were evaluated to be 5 degrees-10 degrees. Possible correlation between conformational changes in the adjacent nucleotides is discussed. PMID:7071023

  7. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    PubMed Central

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans

    2014-01-01

    The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed. PMID:25004977

  8. Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy

    NASA Astrophysics Data System (ADS)

    Bu, Zimei; Farago, Bela; Callaway, David

    2012-02-01

    NHERF1 is a multi-domain scaffolding protein that assembles the signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by a membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model is sufficient to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. The results demonstrate that propagation of allosteric signals to distal sites involves the activation of long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

  9. Structural genomics reveals EVE as a new ASCH/PUA-related domain

    PubMed Central

    Bertonati, Claudia; Punta, Marco; Fischer, Markus; Yachdav, Guy; Forouhar, Farhad; Zhou, Weihong; Kuzin, Alexander P.; Seetharaman, Jayaraman; Abashidze, Mariam; Ramelot, Theresa A.; Kennedy, Michael A.; Cort, John R.; Belachew, Adam; Hunt, John F.; Tong, Liang; Montelione, Gaetano T.; Rost, Burkhard

    2014-01-01

    Summary We report on several proteins recently solved by structural genomics consortia, in particular by the Northeast Structural Genomics consortium (NESG). The proteins considered in this study differ substantially in their sequences but they share a similar structural core, characterized by a pseudobarrel five-stranded beta sheet. This core corresponds to the PUA domain-like architecture in the SCOP database. By connecting sequence information with structural knowledge, we characterize a new subgroup of these proteins that we propose to be distinctly different from previously described PUA domain-like domains such as PUA proper or ASCH. We refer to these newly defined domains as EVE. Although EVE may have retained the ability of PUA domains to bind RNA, the available experimental and computational data suggests that both the details of its molecular function and its cellular function differ from those of other PUA domain-like domains. This study of EVE and its relatives illustrates how the combination of structure and genomics creates new insights by connecting a cornucopia of structures that map to the same evolutionary potential. Primary sequence information alone would have not been sufficient to reveal these evolutionary links. PMID:19191354

  10. Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation

    PubMed Central

    Musayev, Faik N.; Zarate-Perez, Francisco; Bardelli, Martino; Bishop, Clayton; Saniev, Emil F.; Linden, R. Michael; Henckaerts, Els; Escalante, Carlos R.

    2015-01-01

    Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ~20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA. PMID:26314310

  11. Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

    PubMed

    Musayev, Faik N; Zarate-Perez, Francisco; Bardelli, Martino; Bishop, Clayton; Saniev, Emil F; Linden, R Michael; Henckaerts, Els; Escalante, Carlos R

    2015-09-29

    Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.

  12. Structural genomics reveals EVE as a new ASCH/PUA-related domain.

    PubMed

    Bertonati, Claudia; Punta, Marco; Fischer, Markus; Yachdav, Guy; Forouhar, Farhad; Zhou, Weihong; Kuzin, Alexander P; Seetharaman, Jayaraman; Abashidze, Mariam; Ramelot, Theresa A; Kennedy, Michael A; Cort, John R; Belachew, Adam; Hunt, John F; Tong, Liang; Montelione, Gaetano T; Rost, Burkhard

    2009-05-15

    We report on several proteins recently solved by structural genomics consortia, in particular by the Northeast Structural Genomics consortium (NESG). The proteins considered in this study differ substantially in their sequences but they share a similar structural core, characterized by a pseudobarrel five-stranded beta sheet. This core corresponds to the PUA domain-like architecture in the SCOP database. By connecting sequence information with structural knowledge, we characterize a new subgroup of these proteins that we propose to be distinctly different from previously described PUA domain-like domains such as PUA proper or ASCH. We refer to these newly defined domains as EVE. Although EVE may have retained the ability of PUA domains to bind RNA, the available experimental and computational data suggests that both the details of its molecular function and its cellular function differ from those of other PUA domain-like domains. This study of EVE and its relatives illustrates how the combination of structure and genomics creates new insights by connecting a cornucopia of structures that map to the same evolutionary potential. Primary sequence information alone would have not been sufficient to reveal these evolutionary links.

  13. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenolog of animal matrix metalloproteinases

    PubMed Central

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y.; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L.; Potempa, Jan; Gomis-Rüth, F. Xavier

    2010-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52-kDa multidomain protein comprising a central 18-kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analyzed the 3D structure of Kly18 in the absence and presence of Mg2+ or Ca2+, which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg2+ or Ca2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally-characterized non-mammalian MMP is a xenolog co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs. PMID:21166898

  14. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases.

    PubMed

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L; Potempa, Jan; Gomis-Rüth, F Xavier

    2011-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg(2+) or Ca(2+) , which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg(2+) or Ca(2+) binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs.

  15. Developing Prospective Elementary Teachers' Flexibility in the Domain of Proportional Reasoning

    ERIC Educational Resources Information Center

    Berk, Dawn; Taber, Susan B.; Gorowara, Christine Carrino; Poetzl, Christina

    2009-01-01

    Flexibility in the use of mathematics procedures consists of the ability to employ multiple solution methods across a set of problems, solve the same problem using multiple methods, and choose strategically from among methods so as to reduce computational demands. The purpose of this study was to characterize prospective elementary teachers' (n =…

  16. Developing Prospective Elementary Teachers' Flexibility in the Domain of Proportional Reasoning

    ERIC Educational Resources Information Center

    Berk, Dawn; Taber, Susan B.; Gorowara, Christine Carrino; Poetzl, Christina

    2009-01-01

    Flexibility in the use of mathematics procedures consists of the ability to employ multiple solution methods across a set of problems, solve the same problem using multiple methods, and choose strategically from among methods so as to reduce computational demands. The purpose of this study was to characterize prospective elementary teachers' (n =…

  17. Social inequalities in the impact of flexible employment on different domains of psychosocial health

    PubMed Central

    Artazcoz, L.; Benach, J.; Borrell, C.; Cortes, I.

    2005-01-01

    Study objectives: (1) To analyse the impact of flexible employment on mental health and job dissatisfaction; and (2) to examine the constraints imposed by flexible employment on men's and women's partnership formation and people's decision to become parents. For the two objectives the potentially different patterns by sex and social class are explored. Design: Cross sectional health survey. Multiple logistic regression models separated for sex and social class (manual and non-manual workers) and controlling for age were fitted. Four types of contractual arrangements have been considered: permanent, fixed term temporary contract, non-fixed term temporary contract, and no contract. Setting: Catalonia (a region in the north east of Spain). Participants: Salaried workers interviewed in the 2002 Catalonian health survey with no longstanding limiting illness, aged 16–64 (1474 men and 998 women). Main results: Fixed term temporary contracts were not associated with poor mental health status. The impact of other forms of flexible employment on mental health depended on the type of contractual arrangement, sex, and social class and it was restricted to less privileged workers, women, and manual male workers. The impact of flexible employment on living arrangements was higher in men. Among both manual and non-manual male workers, those with fixed term temporary contracts were less likely to have children when married or cohabiting and, additionally, among non-manual male workers they also were more likely to remain single (aOR = 2.35; 95%CI = 1.13 to 4.90). Conclusion: Some forms of temporary contracts are related to adverse health and psychosocial outcomes with different patterns depending on the outcome analysed and on sex and social class. Future research should incorporate variables to capture situations of precariousness associated with flexible employment. PMID:16100314

  18. Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain.

    PubMed Central

    Bui, Q T; Zimmerman, J E; Liu, H; Bonini, N M

    2000-01-01

    The eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it. PMID:10835393

  19. The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

    PubMed Central

    Enggist, Elisabeth; Thöny-Meyer, Linda

    2003-01-01

    CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE. PMID:12813076

  20. Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability.

    PubMed

    Kamerzell, Tim J; Middaugh, C Russell

    2007-08-28

    Despite the well-accepted importance of protein flexibility and dynamics in molecular recognition and conformational stability, our understanding of these relationships is incomplete. Immunoglobulin flexibility is essential for antigen binding and adaptation to diverse molecular shapes and sizes. The inherent flexibility of immunoglobulins also renders these molecules suitable for investigating the possible relationships between protein flexibility and stability. To better understand these inter-relationships, we employ generalized perturbation-based two-dimensional correlation FTIR spectroscopy to monitor the time evolution of H-D exchange of an IgG1 as a function of pH. The differential flexibility of various immunoglobulin regions is described in response to an external perturbation and shown to vary widely. The greatest number of regions with differential exchange rates and, thus differential flexibility, is seen at pH 6. Approximately seven, six, five, and four separate states that exchange with different rates were observed at pH 6, 8, 4, and 2, respectively. The overall distribution of exchange rates calculated from the decays of the integrated Amide I and Amide II areas provides further evidence of multiple regions with differential flexibility. The sequence of events at pH 4 determined from the asynchronous vibrational patterns is of significant interest and suggests protonation of Glu and Asp side chains occurs first and initiates changes in the conformation and flexibility of different sheet and turns structure. A complex inter-relationship between differential regional flexibility and conformational coupling (i.e., cooperativity) initiated by changes in pH influences the stability of this IgG.

  1. Revealing and understanding the behavior of structural domain walls from first principles

    NASA Astrophysics Data System (ADS)

    Iniguez, Jorge

    2015-03-01

    Ferroelectric and ferroelastic domain walls (DWs) are becoming the focus of renewed excitement. Modern experimental techniques permit an unprecedented control on domain structures, and it is now possible to produce materials with a large volume fraction occupied by the DWs themselves. Also, recent experiments show that DWs can display distinct properties not present in the domains, which suggests the possibility of using the walls themselves as the functional material in nano-devices. In this talk I will review recent projects in which we have used theory and first-principles simulation to reveal and explain a variety of DW-related effects. The presentation will include the formation of novel two-dimensional crystals at the DWs of a ferroelastic material, the occurrence of ferroic orders (ferroelectric, ferromagnetic) confined at the DWs of various compounds, and cases in which peculiar (and useful) response and switching properties relie on existence of a multi-domain state. I will also summarize experimental evidence for most of these incredible findings, which clearly ratify domain and domain-wall engineering as a powerful strategy to obtain novel functional nano-materials. // Work done in collaboration with many researchers, the main ones being: J.C. Wojdeł (ICMAB-CSIC), C. Magén (INA at U. Zaragoza), M. Mostovoy (U. Groningen), P. Zubko (U. College London), as well as the groups of Beatriz Noheda (U. Groningen), R. Ramesh (UC Berkeley) and J.-M. Triscone (U. Geneva). Supported by MINECO-Spain.

  2. Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex.

    PubMed

    Reboul, Cyril F; Meyer, Grischa R; Porebski, Benjamin T; Borg, Natalie A; Buckle, Ashley M

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

  3. Epitope Flexibility and Dynamic Footprint Revealed by Molecular Dynamics of a pMHC-TCR Complex

    PubMed Central

    Porebski, Benjamin T.; Borg, Natalie A.; Buckle, Ashley M.

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent ‘snapshots’ and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6–9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into ‘scanning’ mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography. PMID:22412359

  4. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

    PubMed Central

    Smyllie, Nicola J.; Chesham, Johanna E.; Hamnett, Ryan; Maywood, Elizabeth S.; Hastings, Michael H.

    2016-01-01

    The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. PMID:26966234

  5. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions.

    PubMed

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N; Flood, Beverly E; Bailey, Jake V; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  6. Prying open single GroES ring complexes by force reveals cooperativity across domains.

    PubMed

    Ikeda-Kobayashi, Akiko; Taniguchi, Yukinori; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2012-04-18

    Understanding how the mechanical properties of a protein complex emerge from the interplay of intra- and interchain interactions is vital at both fundamental and applied levels. To investigate whether interdomain cooperativity affects protein mechanical strength, we employed single-molecule force spectroscopy to probe the mechanical stability of GroES, a homoheptamer with a domelike quaternary stucture stabilized by intersubunit interactions between the first and last β-strands of adjacent domains. A GroES variant was constructed in which each subunit of the GroES heptamer is covalently linked to adjacent subunits by tripeptide linkers and folded domains of protein L are introduced to the heptamer's termini as handle molecules. The force-distance profiles for GroES unfolding showed, for the first time that we know of, a mechanical phenotype whereby seven distinct force peaks, with alternating behavior of unfolding force and contour length (ΔL(c)), were observed with increasing unfolding-event number. Unfolding of (GroES)(7) is initiated by breakage of the interface between domains 1 and 7 at low force, which imparts a polarity to (GroES)(7) that results in two distinct mechanical phenotypes of these otherwise identical protein domains. Unfolding then proceeds by peeling domains off the domelike native structure by sequential repetition of the denaturation of mechanically weak (unfoldon 1) and strong (unfoldon 2) units. These results indicate that domain-domain interactions help to determine the overall mechanical strength and unfolding pathway of the oligomeric structure. These data reveal an unexpected richness in the mechanical behavior of this homopolyprotein, yielding a complex with greater mechanical strength and properties distinct from those that would be apparent for GroES domains in isolation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Multi-task connectivity reveals flexible hubs for adaptive task control.

    PubMed

    Cole, Michael W; Reynolds, Jeremy R; Power, Jonathan D; Repovs, Grega; Anticevic, Alan; Braver, Todd S

    2013-09-01

    Extensive evidence suggests that the human ability to adaptively implement a wide variety of tasks is preferentially a result of the operation of a fronto-parietal brain network (FPN). We hypothesized that this network's adaptability is made possible by flexible hubs: brain regions that rapidly update their pattern of global functional connectivity according to task demands. Using recent advances in characterizing brain network organization and dynamics, we identified mechanisms consistent with the flexible hub theory. We found that the FPN's brain-wide functional connectivity pattern shifted more than those of other networks across a variety of task states and that these connectivity patterns could be used to identify the current task. Furthermore, these patterns were consistent across practiced and novel tasks, suggesting that reuse of flexible hub connectivity patterns facilitates adaptive (novel) task performance. Together, these findings support a central role for fronto-parietal flexible hubs in cognitive control and adaptive implementation of task demands.

  8. Structure analysis reveals the flexibility of the ADAMTS-5 active site

    SciTech Connect

    Shieh, Huey-Sheng; Tomasselli, Alfredo G.; Mathis, Karl J.; Schnute, Mark E.; Woodard, Scott S.; Caspers, Nicole; Williams, Jennifer M.; Kiefer, James R.; Munie, Grace; Wittwer, Arthur; Malfait, Anne-Marie; Tortorella, Micky D.

    2012-03-02

    A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.

  9. Efficiency and Flexibility of Indirect Addition in the Domain of Multi-Digit Subtraction

    ERIC Educational Resources Information Center

    Torbeyns, Joke; Ghesquiere, Pol; Verschaffel, Lieven

    2009-01-01

    This article discusses the characteristics of the indirect addition strategy (IA) in the domain of multi-digit subtraction. In two studies, adults' use of IA on three-digit subtractions with a small, medium, or large difference between the integers was analysed using the choice/no-choice method. Results from both studies indicate that adults…

  10. Ion mobility-mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility

    NASA Astrophysics Data System (ADS)

    Zhou, Min; Politis, Argyris; Davies, Roberta B.; Liko, Idlir; Wu, Kuan-Jung; Stewart, Alastair G.; Stock, Daniela; Robinson, Carol V.

    2014-03-01

    Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.

  11. A comparative analysis of the foamy and ortho virus capsid structures reveals an ancient domain duplication.

    PubMed

    Taylor, William R; Stoye, Jonathan P; Taylor, Ian A

    2017-04-04

    The Spumaretrovirinae (foamy viruses) and the Orthoretrovirinae (e.g. HIV) share many similarities both in genome structure and the sequences of the core viral encoded proteins, such as the aspartyl protease and reverse transcriptase. Similarity in the gag region of the genome is less obvious at the sequence level but has been illuminated by the recent solution of the foamy virus capsid (CA) structure. This revealed a clear structural similarity to the orthoretrovirus capsids but with marked differences that left uncertainty in the relationship between the two domains that comprise the structure. We have applied protein structure comparison methods in order to try and resolve this ambiguous relationship. These included both the DALI method and the SAP method, with rigorous statistical tests applied to the results of both methods. For this, we employed collections of artificial fold 'decoys' (generated from the pair of native structures being compared) to provide a customised background distribution for each comparison, thus allowing significance levels to be estimated. We have shown that the relationship of the two domains conforms to a simple linear correspondence rather than a domain transposition. These similarities suggest that the origin of both viral capsids was a common ancestor with a double domain structure. In addition, we show that there is also a significant structural similarity between the amino and carboxy domains in both the foamy and ortho viruses. These results indicate that, as well as the duplication of the double domain capsid, there may have been an even more ancient gene-duplication that preceded the double domain structure. In addition, our structure comparison methodology demonstrates a general approach to problems where the components have a high intrinsic level of similarity.

  12. Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

    PubMed Central

    Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

    2016-01-01

    Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks. PMID:26935805

  13. Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

    NASA Astrophysics Data System (ADS)

    Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

    2016-03-01

    Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

  14. Revealing a new activity of the human Dicer DUF283 domain in vitro

    PubMed Central

    Kurzynska-Kokorniak, Anna; Pokornowska, Maria; Koralewska, Natalia; Hoffmann, Weronika; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek

    2016-01-01

    The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs. PMID:27045313

  15. Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots.

    PubMed

    Springer, Nathan M; Napoli, Carolyn A; Selinger, David A; Pandey, Ritu; Cone, Karen C; Chandler, Vicki L; Kaeppler, Heidi F; Kaeppler, Shawn M

    2003-06-01

    Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

  16. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions

    PubMed Central

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming “Candidatus Thiomargarita nelsonii Thio36”, and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. “Ca. T. nelsonii Thio36” is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that “Ca. T. nelsonii Thio36” can function as a chemolithoautotroph. Carbon can be fixed via the Calvin–Benson–Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, “Ca. T. nelsonii Thio36” also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae “Ca. T. nelsonii Thio36” encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of “Ca. T. nelsonii Thio36” provides additional insight into the ecology of

  17. Single-cell sequencing of Thiomargarita reveals genomic flexibility for adaptation to dynamic redox conditions

    DOE PAGES

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; ...

    2016-06-21

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiornargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of amore » chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In conclusion, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  18. Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

    SciTech Connect

    S Menon; S Wang

    2011-12-31

    The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, the switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.

  19. Conformational flexibility of a human immunoglobulin light chain variable domain by relaxation dispersion nuclear magnetic resonance spectroscopy: implications for protein misfolding and amyloid assembly.

    PubMed

    Mukherjee, Sujoy; Pondaven, Simon P; Jaroniec, Christopher P

    2011-07-05

    The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide (15)N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, (15)N Carr--Purcell--Meiboom--Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5-20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ~2-4% range. Upon acidification, the minor conformer populations increase significantly, to ~10-15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.

  20. C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain.

    PubMed

    Zhou, Yan-Feng; Nan, Beiyan; Nan, Jie; Ma, Qingjun; Panjikar, Santosh; Liang, Yu-He; Wang, Yiping; Su, Xiao-Dong

    2008-10-31

    C(4)-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C(4)-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C(4)-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C(4) succinate, and a complex with C(3) malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C(4)-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.

  1. Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

    PubMed

    Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo; Schmidt, Sven H; Sankaran, Banumathi; Pavlidis, Ioannis V; Kim, Choel; Herberg, Friedrich W

    2017-07-06

    Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  2. Multi-task connectivity reveals flexible hubs for adaptive task control

    PubMed Central

    Cole, Michael W.; Reynolds, Jeremy R.; Power, Jonathan D.; Repovs, Grega; Anticevic, Alan; Braver, Todd S.

    2013-01-01

    Extensive evidence suggests the human ability to adaptively implement a wide variety of tasks is preferentially due to the operation of a fronto-parietal brain network. We hypothesized that this network’s adaptability is made possible by ‘flexible hubs’ – brain regions that rapidly update their pattern of global functional connectivity according to task demands. We utilized recent advances in characterizing brain network organization and dynamics to identify mechanisms consistent with the flexible hub theory. We found that the fronto-parietal network’s brain-wide functional connectivity pattern shifted more than other networks’ across a variety of task states, and that these connectivity patterns could be used to identify the current task. Further, these patterns were consistent across practiced and novel tasks, suggesting reuse of flexible hub connectivity patterns facilitates adaptive (novel) task performance. Together, these findings support a central role for fronto-parietal flexible hubs in cognitive control and adaptive implementation of task demands generally. PMID:23892552

  3. Crystal structure of the HCV IRES central domain reveals strategy for start-codon positioning.

    PubMed

    Berry, Katherine E; Waghray, Shruti; Mortimer, Stefanie A; Bai, Yun; Doudna, Jennifer A

    2011-10-12

    Translation of hepatitis C viral proteins requires an internal ribosome entry site (IRES) located in the 5' untranslated region of the viral mRNA. The core domain of the hepatitis C virus (HCV) IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. This domain is required for positioning the mRNA start codon correctly on the 40S ribosomal subunit during translation initiation. Here, we present the crystal structure of this RNA, revealing a complex double-pseudoknot fold that establishes the alignment of two helical elements on either side of the four-helix junction. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S ribosomal subunit. This structure, representing the last major domain of HCV-like IRESs to be determined at near-atomic resolution, provides the basis for a comprehensive cryoelectron microscopy-guided model of the intact HCV IRES and its interaction with 40S ribosomal subunits.

  4. Revealing crystalline domains in a mollusc shell single-crystalline prism

    NASA Astrophysics Data System (ADS)

    Mastropietro, F.; Godard, P.; Burghammer, M.; Chevallard, C.; Daillant, J.; Duboisset, J.; Allain, M.; Guenoun, P.; Nouet, J.; Chamard, V.

    2017-09-01

    Biomineralization integrates complex processes leading to an extraordinary diversity of calcareous biomineral crystalline architectures, in intriguing contrast with the consistent presence of a sub-micrometric granular structure. Hence, gaining access to the crystalline architecture at the mesoscale, that is, over a few granules, is key to building realistic biomineralization scenarios. Here we provide the nanoscale spatial arrangement of the crystalline structure within the `single-crystalline' prisms of the prismatic layer of a Pinctada margaritifera shell, exploiting three-dimensional X-ray Bragg ptychography microscopy. We reveal the details of the mesocrystalline organization, evidencing a crystalline coherence extending over a few granules. We additionally prove the existence of larger iso-oriented crystalline domains, slightly misoriented with respect to each other, around one unique rotation axis, and whose shapes are correlated with iso-strain domains. The highlighted mesocrystalline properties support recent biomineralization models involving partial fusion of oriented nanoparticle assembly and/or liquid droplet precursors.

  5. Revealing crystalline domains in a mollusc shell single-crystalline prism.

    PubMed

    Mastropietro, F; Godard, P; Burghammer, M; Chevallard, C; Daillant, J; Duboisset, J; Allain, M; Guenoun, P; Nouet, J; Chamard, V

    2017-09-01

    Biomineralization integrates complex processes leading to an extraordinary diversity of calcareous biomineral crystalline architectures, in intriguing contrast with the consistent presence of a sub-micrometric granular structure. Hence, gaining access to the crystalline architecture at the mesoscale, that is, over a few granules, is key to building realistic biomineralization scenarios. Here we provide the nanoscale spatial arrangement of the crystalline structure within the 'single-crystalline' prisms of the prismatic layer of a Pinctada margaritifera shell, exploiting three-dimensional X-ray Bragg ptychography microscopy. We reveal the details of the mesocrystalline organization, evidencing a crystalline coherence extending over a few granules. We additionally prove the existence of larger iso-oriented crystalline domains, slightly misoriented with respect to each other, around one unique rotation axis, and whose shapes are correlated with iso-strain domains. The highlighted mesocrystalline properties support recent biomineralization models involving partial fusion of oriented nanoparticle assembly and/or liquid droplet precursors.

  6. Origins of Structural Flexibility in Protein-Based Supramolecular Polymers Revealed by DEER Spectroscopy

    PubMed Central

    2015-01-01

    Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer. PMID:25060334

  7. Structure of the flexible amino terminal domain of prion protein bound to a sulfated glycan

    PubMed Central

    Taubner, Lara M.; Bienkiewicz, Ewa A.; Copié, Valérie; Caughey, Byron

    2010-01-01

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence PHGGGWGQ that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (1H, 13C, 15N) nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23–106 (PrP 23–106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23–106 remains disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data, which demonstrate that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan sidechains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23–106 when bound to PPS, consistent with the alignment of tryptophan sidechains. Chemical shift mapping identified binding sites on PrP 23–106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. PMID:19913031

  8. Structure of the flexible amino-terminal domain of prion protein bound to a sulfated glycan.

    PubMed

    Taubner, Lara M; Bienkiewicz, Ewa A; Copié, Valérie; Caughey, Byron

    2010-01-22

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear ((1)H, (13)C, (15)N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The beta-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. Published by Elsevier Ltd.

  9. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    SciTech Connect

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N.

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  10. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  11. Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

    PubMed Central

    Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

    2016-01-01

    The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

  12. Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners.

    PubMed

    Gemperle, Jakub; Hexnerová, Rozálie; Lepšík, Martin; Tesina, Petr; Dibus, Michal; Novotný, Marian; Brábek, Jan; Veverka, Václav; Rosel, Daniel

    2017-08-14

    CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.

  13. Structure of the Spt16 Middle Domain Reveals Functional Features of the Histone Chaperone FACT*

    PubMed Central

    Kemble, David J.; Whitby, Frank G.; Robinson, Howard; McCullough, Laura L.; Formosa, Tim; Hill, Christopher P.

    2013-01-01

    The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins. PMID:23417676

  14. Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging.

    PubMed

    Frankel, D J; Pfeiffer, J R; Surviladze, Z; Johnson, A E; Oliver, J M; Wilson, B S; Burns, A R

    2006-04-01

    Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging were used to observe in aqueous buffer the three-dimensional landscape of the inner surface of membrane sheets stripped from fixed tumor mast cells. The AFM images reveal prominent, irregularly shaped raised domains that label with fluorescent markers for both resting and activated immunoglobin E receptors (FcepsilonRI), as well as with cholera toxin-aggregated GM1 and clathrin. The latter suggests that coated pits bud from these regions. These features are interspersed with flatter regions of membrane and are frequently surrounded and interconnected by cytoskeletal assemblies. The raised domains shrink in height by approximately 50% when cholesterol is extracted with methyl-beta-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterol-enriched dark patches observed in transmission electron microscopy (TEM). These patches were previously identified as sites of signaling and endocytosis based on their localization of activated FcepsilonRI, at least 10 associated signaling molecules, and the presence of clathrin-coated pits. Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton.

  15. STRUCTURE OF THE DNA REPAIR HELICASE HEL308 REVEALS DNA BINDING AND AUTOINHIBITORY DOMAINS

    PubMed Central

    Richards, Jodi; Johnson, Ken; Liu, Huanting; Oke, Stephen McMahon. Muse; Carter, Lester; Naismith, James H; White, Malcolm F

    2012-01-01

    Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest, and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high-resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as a molecular brake, clamping the ssDNA extruded through the central pore of the helicase structure to limit the enzyme’s helicase activity. This provides an elegant mechanism to tune the enzyme’s processivity to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, suggesting that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates. PMID:18056710

  16. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    PubMed Central

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard

    2016-01-01

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopox-viruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. PMID:23816434

  17. Comparative Analysis of RNA Families Reveals Distinct Repertoires for Each Domain of Life

    PubMed Central

    Hoeppner, Marc P.; Gardner, Paul P.; Poole, Anthony M.

    2012-01-01

    The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner. PMID:23133357

  18. Comparative analysis of RNA families reveals distinct repertoires for each domain of life.

    PubMed

    Hoeppner, Marc P; Gardner, Paul P; Poole, Anthony M

    2012-01-01

    The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner.

  19. Protein Domain Analysis of Genomic Sequence Data Reveals Regulation of LRR Related Domains in Plant Transpiration in Ficus

    PubMed Central

    Lang, Tiange; Yin, Kangquan; Liu, Jinyu; Cao, Kunfang; Cannon, Charles H.; Du, Fang K.

    2014-01-01

    Predicting protein domains is essential for understanding a protein’s function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion. PMID:25269070

  20. A systematic, family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions

    PubMed Central

    Chang, Bryan H.; Gujral, Taranjit S.; Karp, Ethan S.; BuKhalid, Raghida; Grantcharova, Viara P.; MacBeath, Gavin

    2012-01-01

    Summary PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C-termini of their target proteins. In a few instances, however, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify novel interactions and co-affinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand. PMID:21944753

  1. Endpoint Force Fluctuations Reveal Flexible Rather Than Synergistic Patterns of Muscle Cooperation

    PubMed Central

    Kutch, Jason J.; Kuo, Arthur D.; Bloch, Anthony M.; Rymer, William Z.

    2008-01-01

    We developed a new approach to investigate how the nervous system activates multiple redundant muscles by studying the endpoint force fluctuations during isometric force generation at a multi-degree-of-freedom joint. We hypothesized that, due to signal-dependent muscle force noise, endpoint force fluctuations would depend on the target direction of index finger force and that this dependence could be used to distinguish flexible from synergistic activation of the musculature. We made high-gain measurements of isometric forces generated to different target magnitudes and directions, in the plane of index finger metacarpophalangeal joint abduction–adduction/flexion–extension. Force fluctuations from each target were used to calculate a covariance ellipse, the shape of which varied as a function of target direction. Directions with narrow ellipses were approximately aligned with the estimated mechanical actions of key muscles. For example, targets directed along the mechanical action of the first dorsal interosseous (FDI) yielded narrow ellipses, with 88% of the variance directed along those target directions. It follows the FDI is likely a prime mover in this target direction and that, at most, 12% of the force variance could be explained by synergistic coupling with other muscles. In contrast, other target directions exhibited broader covariance ellipses with as little as 30% of force variance directed along those target directions. This is the result of cooperation among multiple muscles, based on independent electromyographic recordings. However, the pattern of cooperation across target directions indicates that muscles are recruited flexibly in accordance with their mechanical action, rather than in fixed groupings. PMID:18799603

  2. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

    PubMed Central

    Gómez-Marín, Carlos; Tena, Juan J.; Acemel, Rafael D.; López-Mayorga, Macarena; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Maeso, Ignacio; Beccari, Leonardo; Aneas, Ivy; Vielmas, Erika; Bovolenta, Paola; Nobrega, Marcelo A.; Carvajal, Jaime; Gómez-Skarmeta, José Luis

    2015-01-01

    Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution. PMID:26034287

  3. Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome.

    PubMed

    Vieweger, Mario; Holmstrom, Erik D; Nesbitt, David J

    2015-12-15

    Metabolite-dependent conformational switching in RNA riboswitches is now widely accepted as a critical regulatory mechanism for gene expression in bacterial systems. More recently, similar gene regulation mechanisms have been found to be important for viral systems as well. One of the most abundant and best-studied systems is the tRNA-like structure (TLS) domain, which has been found to occur in many plant viruses spread across numerous genera. In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques. In particular, burst fluorescence methods are exploited to observe metal-ion ([M(n+)])-induced folding in freely diffusing RNA constructs resembling the minimal TLS element of brome mosaic virus RNA3. The results of these experiments reveal a complex equilibrium of at least three distinct populations. A stepwise, or consecutive, thermodynamic model for TLS folding is developed, which is in good agreement with the [M(n+)]-dependent evolution of conformational populations and existing structural information in the literature. Specifically, this folding pathway explains the metal-ion dependent formation of a functional TLS domain from unfolded RNAs via two consecutive steps: 1) hybridization of a long-range stem interaction, followed by 2) formation of a 3'-terminal pseudoknot. These two conformational transitions are well described by stepwise dissociation constants for [Mg(2+)] (K1 = 328 ± 30 μM and K2 = 1092 ± 183 μM) and [Na(+)] (K1 = 74 ± 6 mM and K2 = 243 ± 52 mM)-induced folding. The proposed thermodynamic model is further supported by inhibition studies of the long-range stem interaction using a complementary DNA oligomer, which effectively shifts the dynamic equilibrium toward the unfolded conformation. Implications of this multistep conformational folding mechanism are discussed with regard to regulation of virus replication.

  4. Flexible and scalable methods for quantifying stochastic variability in the era of massive time-domain astronomical data sets

    SciTech Connect

    Kelly, Brandon C.; Becker, Andrew C.; Sobolewska, Malgosia; Siemiginowska, Aneta; Uttley, Phil

    2014-06-10

    We present the use of continuous-time autoregressive moving average (CARMA) models as a method for estimating the variability features of a light curve, and in particular its power spectral density (PSD). CARMA models fully account for irregular sampling and measurement errors, making them valuable for quantifying variability, forecasting and interpolating light curves, and variability-based classification. We show that the PSD of a CARMA model can be expressed as a sum of Lorentzian functions, which makes them extremely flexible and able to model a broad range of PSDs. We present the likelihood function for light curves sampled from CARMA processes, placing them on a statistically rigorous foundation, and we present a Bayesian method to infer the probability distribution of the PSD given the measured light curve. Because calculation of the likelihood function scales linearly with the number of data points, CARMA modeling scales to current and future massive time-domain data sets. We conclude by applying our CARMA modeling approach to light curves for an X-ray binary, two active galactic nuclei, a long-period variable star, and an RR Lyrae star in order to illustrate their use, applicability, and interpretation.

  5. Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

    PubMed

    Zhao, Yunjie; Zeng, Chen; Massiah, Michael A

    2015-01-01

    The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

  6. Short LOV Proteins in Methylocystis Reveal Insight into LOV Domain Photocycle Mechanisms

    PubMed Central

    El-Arab, Kaley K.; Pudasaini, Ashutosh; Zoltowski, Brian D.

    2015-01-01

    Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in Methylocystis. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins. PMID:25933162

  7. Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor

    SciTech Connect

    Mirza, I.; Yachnin, B; Wang, S; Grosse, S; Bergeron, H; Imura, A; Iwaki, H; Hasegawa, Y; Lau, P; Berghuis, A

    2009-01-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O{sub 2} as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 {angstrom}. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  8. Impaired neurodevelopment by the low complexity domain of CPEB4 reveals a convergent pathway with neurodegeneration

    PubMed Central

    Shin, Jihae; Salameh, Johnny S.; Richter, Joel D.

    2016-01-01

    CPEB4 is an RNA binding protein expressed in neuronal tissues including brain and spinal cord. CPEB4 has two domains: one that is structured for RNA binding and one that is unstructured and low complexity that has no known function. Unstructured low complexity domains (LCDs) in proteins are often found in RNA-binding proteins and have been implicated in motor neuron degenerative diseases such as amyotrophic lateral sclerosis, indicating that these regions mediate normal RNA processing as well as pathological events. While CPEB4 null knockout mice are normal, animals expressing only the CPEB4 LCD are neonatal lethal with impaired mobility that display defects in neuronal development such as reduced motor axon branching and abnormal neuromuscular junction formation. Although full-length CPEB4 is nearly exclusively cytoplasmic, the CPEB4 LCD forms nucleolar aggregates and CPEB4 LCD-expressing animals have altered ribosomal RNA biogenesis, ribosomal protein gene expression, and elevated levels of stress response genes such as the actin-bundling protein DRR1, which impedes neurite outgrowth. Some of these features share similarities with other LCD-related neurodegenerative disease. Most strikingly, DRR1 appears to be a common focus of several neurodevelopmental and neurodegenerative disorders. Our study reveals a possible molecular convergence between a neurodevelopmental defect and neurodegeneration mediated by LCDs. PMID:27381259

  9. Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

    PubMed

    Mirza, I Ahmad; Yachnin, Brahm J; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K; Berghuis, Albert M

    2009-07-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  10. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC.

    PubMed

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N; Pandey, Akhilesh

    2011-03-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain-containing 1A; gene symbol ANKS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    PubMed Central

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N.; Pandey, Akhilesh

    2011-01-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain containing 1A; gene symbol AKNS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. PMID:21081186

  12. Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.

    PubMed

    Sauvage, Eric; Herman, Raphaël; Petrella, Stephanie; Duez, Colette; Bouillenne, Fabrice; Frère, Jean-Marie; Charlier, Paulette

    2005-09-02

    Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.

  13. Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

    PubMed

    Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

    2016-04-01

    DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions. We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2-NuRD. Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo.

  14. Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions

    PubMed Central

    Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

    2016-01-01

    Aim: DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2–NuRD (nucleosome remodeling deacetylase) interactions. Experimental procedures: We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2–NuRD. Results: Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Conclusion: Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo. PMID:27066839

  15. Repeat Tracking of Individual Songbirds Reveals Consistent Migration Timing but Flexibility in Route

    PubMed Central

    Fraser, Kevin C.; McKinnon, Emily A.; Stutchbury, Bridget J. M.

    2012-01-01

    Tracking repeat migratory journeys of individual animals is required to assess phenotypic plasticity of individual migration behaviour in space and time. We used light-level geolocators to track the long-distance journeys of migratory songbirds (wood thrush, Hylocichla mustelina), and, for the first time, repeat journeys of individuals. We compare between- and within-individual variation in migration to examine flexibility of timing and route in spring and autumn. Date of departure from wintering sites in Central America, along with sex and age factors, explained most of the variation (71%) in arrival date at North American breeding sites. Spring migration showed high within-individual repeatability in timing, but not in route. In particular, spring departure dates of individuals were highly repeatable, with a mean difference between years of just 3 days. Autumn migration timing and routes were not repeatable. Our results provide novel evidence of low phenotypic plasticity in timing of spring migration, which may limit the ability of individuals to adjust migration schedules in response to climate change. PMID:22848395

  16. Structural analysis reveals the flexible C-terminus of Nop15 undergoes rearrangement to recognize a pre-ribosomal RNA folding intermediate.

    PubMed

    Zhang, Jun; Gonzalez, Lauren E; Hall, Traci M Tanaka

    2017-03-17

    The RNA recognition motif (RRM) is the most abundant RNA-binding domain in eukaryotes, and it plays versatile roles in RNA metabolism. Despite its abundance, diversity of RRM structure and function is generated by variations on a conserved core. Yeast Nop15 is an RRM protein that is essential for large ribosomal subunit biogenesis. We determined a 2.0 Å crystal structure of Nop15 that reveals a C-terminal α-helical region obscures its canonical RNA-binding surface. Small-angle X-ray scattering, NMR and RNA-binding analyses further reveal that the C-terminal residues of Nop15 are highly flexible, but essential for tight RNA binding. Moreover, comparison with a recently reported cryo-electron microscopy structure indicates that dramatic rearrangement of the C-terminal region of Nop15 in the pre-ribosome exposes the RNA-binding surface to recognize the base of its stem-loop target RNA and extends a newly-formed α helix to the distal loop where it forms protein interactions. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

  17. Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains

    PubMed Central

    Dattoli, Ada A.; Hink, Mark A.; DuBuc, Timothy Q.; Teunisse, Bram J.; Goedhart, Joachim; Röttinger, Eric; Postma, Marten

    2015-01-01

    SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus. PMID:26190255

  18. Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

    PubMed

    Dattoli, Ada A; Hink, Mark A; DuBuc, Timothy Q; Teunisse, Bram J; Goedhart, Joachim; Röttinger, Eric; Postma, Marten

    2015-07-20

    SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

  19. Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

    PubMed Central

    Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

    2006-01-01

    Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

  20. Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

    SciTech Connect

    Dubini, A.; Mus, F.; Seibert, M.; Grossman, A. R.; Posewitz, M. C.

    2009-03-13

    The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

  1. Experimental demonstration of an OpenFlow based software-defined optical network employing packet, fixed and flexible DWDM grid technologies on an international multi-domain testbed.

    PubMed

    Channegowda, M; Nejabati, R; Rashidi Fard, M; Peng, S; Amaya, N; Zervas, G; Simeonidou, D; Vilalta, R; Casellas, R; Martínez, R; Muñoz, R; Liu, L; Tsuritani, T; Morita, I; Autenrieth, A; Elbers, J P; Kostecki, P; Kaczmarek, P

    2013-03-11

    Software defined networking (SDN) and flexible grid optical transport technology are two key technologies that allow network operators to customize their infrastructure based on application requirements and therefore minimizing the extra capital and operational costs required for hosting new applications. In this paper, for the first time we report on design, implementation & demonstration of a novel OpenFlow based SDN unified control plane allowing seamless operation across heterogeneous state-of-the-art optical and packet transport domains. We verify and experimentally evaluate OpenFlow protocol extensions for flexible DWDM grid transport technology along with its integration with fixed DWDM grid and layer-2 packet switching.

  2. Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

    PubMed

    White, A P; Collinson, S K; Banser, P A; Dolhaine, D J; Kay, W W

    2000-02-18

    Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies

  3. Structure of USP7 catalytic domain and three Ubl-domains reveals a connector α-helix with regulatory role.

    PubMed

    Kim, Robbert Q; van Dijk, Willem J; Sixma, Titia K

    2016-07-01

    Ubiquitin conjugation is an important signal in cellular pathways, changing the fate of a target protein, by degradation, relocalisation or complex formation. These signals are balanced by deubiquitinating enzymes (DUBs), which antagonize ubiquitination of specific protein substrates. Because ubiquitination pathways are critically important, DUB activity is often carefully controlled. USP7 is a highly abundant DUB with numerous targets that plays complex roles in diverse pathways, including DNA regulation, p53 stress response and endosomal protein recycling. Full-length USP7 switches between an inactive and an active state, tuned by the positioning of 5 Ubl folds in the C-terminal HUBL domain. The active state requires interaction between the last two Ubls (USP7(45)) and the catalytic domain (USP7(CD)), and this can be promoted by allosteric interaction from the first 3 Ubl domains of USP7 (USP7(123)) interacting with GMPS. Here we study the transition between USP7 states. We provide a crystal structure of USP7(CD123) and show that CD and Ubl123 are connected via an extended charged alpha helix. Mutational analysis is used to determine whether the charge and rigidity of this 'connector helix' are important for full USP7 activity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Genome-wide search for eliminylating domains reveals novel function for BLES03-like proteins.

    PubMed

    Khater, Shradha; Mohanty, Debasisa

    2014-07-24

    Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homologs in eukaryotes was carried out. Our fold-based search revealed structural and catalytic site similarity between bacterial phosphothreonine lyases and BLES03 (basophilic leukemia-expressed protein 03), a human protein with unknown function. Ligand induced conformational changes similar to bacterial phosphothreonine lyases, and movement of crucial arginines in the loop region to the catalytic pocket upon binding of phosphothreonine-containing peptides was seen during docking and molecular dynamics studies. Genome-wide search for BLES03 homologs using sensitive profile-based methods revealed their presence not only in eukaryotic classes such as chordata and fungi but also in bacterial and archaebacterial classes. The synteny of these archaebacterial BLES03-like proteins was remarkably similar to that of type IV lantibiotic synthetases which harbor LanL-like phosphothreonine lyase domains. Hence, context-based analysis reinforced our earlier sequence/structure-based prediction of phosphothreonine lyase catalytic function for BLES03. Our in silico analysis has revealed that BLES03-like proteins with previously unknown function are novel eukaryotic phosphothreonine lyases involved in biosynthesis of dehydro amino acids, whereas their bacterial and archaebacterial counterparts might be involved in biosynthesis of natural products similar to lantibiotics.

  5. Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression

    PubMed Central

    Libbrecht, Maxwell W.; Ay, Ferhat; Hoffman, Michael M.; Gilbert, David M.; Bilmes, Jeffrey A.; Noble, William Stafford

    2015-01-01

    The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term “specific expression domains.” We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. PMID:25677182

  6. Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression.

    PubMed

    Libbrecht, Maxwell W; Ay, Ferhat; Hoffman, Michael M; Gilbert, David M; Bilmes, Jeffrey A; Noble, William Stafford

    2015-04-01

    The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term "specific expression domains." We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. © 2015 Libbrecht et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Bilayer deformation by the Kv channel voltage sensor domain revealed by self-assembly simulations.

    PubMed

    Bond, Peter J; Sansom, Mark S P

    2007-02-20

    Coarse-grained molecular dynamics simulations are used to explore the interaction with a phospholipid bilayer of the voltage sensor (VS) domain and the S4 helix from the archaebacterial voltage-gated potassium (Kv) channel KvAP. Multiple 2-mus self-assembly simulations reveal that the isolated S4 helix may adopt either interfacial or transmembrane (TM) locations with approximately equal probability. In the TM state, the insertion of the voltage-sensing region of S4 is facilitated via local bilayer deformation that, combined with side chain "snorkeling," enables its Arg side chains to interact with lipid headgroups and water. Multiple 0.2-mus self-assembly simulations of the VS domain are also performed, along with simulations of MscL and KcsA, to permit comparison with more "canonical" integral membrane protein structures. All three stably adopt a TM orientation within a bilayer. For MscL and KcsA, there is no significant bilayer deformation. In contrast, for the VS, there is considerable local deformation, which is again primarily due to the lipid-exposed S4. It is shown that for both the VS and isolated S4 helix, the positively charged side chains of S4 are accommodated within the membrane through a combination of stabilizing interactions with lipid glycerol and headgroup regions, water, and anionic side chains. Our results support the possibility that bilayer deformation around key gating charge residues in Kv channels may result in "focusing" of the electrostatic field, and indicate that, when considering competing models of voltage-sensing, it is essential to consider the dynamics and structure of not only the protein but also of the local lipid environment.

  8. Thickness dependence of magnetic anisotropy and domains in amorphous Co40Fe40B20 thin films grown on PET flexible substrates

    NASA Astrophysics Data System (ADS)

    Tang, Zhenhua; Ni, Hao; Lu, Biao; Zheng, Ming; Huang, Yong-An; Lu, Sheng-Guo; Tang, Minghua; Gao, Ju

    2017-03-01

    The amorphous Co40Fe40B20 (CoFeB) films (5-200 nm in thickness) were grown on flexible polyethylene terephthalate (PET) substrates using the DC magnetron-sputtering method. The thickness dependence of structural and magnetic properties of flexible CoFeB thin films was investigated in detail. The in-plane uniaxial magnetic anisotropy induced by strain as a function of thickness was obtained in flexible CoFeB thin films, and a critical thickness of 150 nm for in-plane magnetic anisotropy was observed. Moreover, the domains and the uniaxial anisotropy as a function of angular direction of applied magnetic field were characterized. The results show potential for designing CoFeB-based flexible spintronic devices in which the physical parameters could be tailored by controlling the thickness of the thin film.

  9. Flexibility and Stability in Sensory Processing Revealed Using Visual-to-Auditory Sensory Substitution

    PubMed Central

    Hertz, Uri; Amedi, Amir

    2015-01-01

    The classical view of sensory processing involves independent processing in sensory cortices and multisensory integration in associative areas. This hierarchical structure has been challenged by evidence of multisensory responses in sensory areas, and dynamic weighting of sensory inputs in associative areas, thus far reported independently. Here, we used a visual-to-auditory sensory substitution algorithm (SSA) to manipulate the information conveyed by sensory inputs while keeping the stimuli intact. During scan sessions before and after SSA learning, subjects were presented with visual images and auditory soundscapes. The findings reveal 2 dynamic processes. First, crossmodal attenuation of sensory cortices changed direction after SSA learning from visual attenuations of the auditory cortex to auditory attenuations of the visual cortex. Secondly, associative areas changed their sensory response profile from strongest response for visual to that for auditory. The interaction between these phenomena may play an important role in multisensory processing. Consistent features were also found in the sensory dominance in sensory areas and audiovisual convergence in associative area Middle Temporal Gyrus. These 2 factors allow for both stability and a fast, dynamic tuning of the system when required. PMID:24518756

  10. Simulations reveal the role of composition into the atomic-level flexibility of bioactive glass cements.

    PubMed

    Tian, Kun Viviana; Chass, Gregory A; Di Tommaso, Devis

    2016-01-14

    Bioactive glass ionomer cements (GICs), the reaction product of a fluoro-alumino-silicate glass and polyacrylic acid, have been in effective use in dentistry for over 40 years and more recently in orthopaedics and medical implantation. Their desirable properties have affirmed GIC's place in the medical materials community, yet are limited to non-load bearing applications due to the brittle nature of the hardened composite cement, thought to arise from the glass component and the interfaces it forms. Towards helping resolve the fundamental bases of the mechanical shortcomings of GICs, we report the 1st ever computational models of a GIC-relevant component. Ab initio molecular dynamics simulations were employed to generate and characterise three fluoro-alumino-silicate glasses of differing compositions with focus on resolving the atomic scale structural and dynamic contributions of aluminium, phosphorous and fluorine. Analyses of the glasses revealed rising F-content leading to the expansion of the glass network, compression of Al-F bonding, angular constraint at Al-pivots, localisation of alumino-phosphates and increased fluorine diffusion. Together, these changes to the structure, speciation and dynamics with raised fluorine content impart an overall rigidifying effect on the glass network, and suggest a predisposition to atomic-level inflexibility, which could manifest in the ionomer cements they form.

  11. Systematic analyses reveal uniqueness and origin of the CFEM domain in fungi

    PubMed Central

    Zhang, Zhen-Na; Wu, Qin-Yi; Zhang, Gui-Zhi; Zhu, Yue-Yan; Murphy, Robert W.; Liu, Zhen; Zou, Cheng-Gang

    2015-01-01

    CFEM domain commonly occurs in fungal extracellular membrane proteins. To provide insights for understanding putative functions of CFEM, we investigate the evolutionary dynamics of CFEM domains by systematic comparative genomic analyses among diverse animals, plants, and more than 100 fungal species, which are representative across the entire group of fungi. We here show that CFEM domain is unique to fungi. Experiments using tissue culture demonstrate that the CFEM-containing ESTs in some plants originate from endophytic fungi. We also find that CFEM domain does not occur in all fungi. Its single origin dates to the most recent common ancestors of Ascomycota and Basidiomycota, instead of multiple origins. Although the length and architecture of CFEM domains are relatively conserved, the domain-number varies significantly among different fungal species. In general, pathogenic fungi have a larger number of domains compared to other species. Domain-expansion across fungal genomes appears to be driven by domain duplication and gene duplication via recombination. These findings generate a clear evolutionary trajectory of CFEM domains and provide novel insights into the functional exchange of CFEM-containing proteins from cell-surface components to mediators in host-pathogen interactions. PMID:26255557

  12. Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

    PubMed Central

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S.; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  13. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases.

    PubMed

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

  14. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

    PubMed Central

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements. PMID:26024355

  15. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

    PubMed

    Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

    2013-02-01

    Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

  16. A Naturally-Occurring Transcript Variant of MARCO Reveals the SRCR Domain is Critical for Function

    PubMed Central

    Novakowski, Kyle E.; Huynh, Angela; Han, SeongJun; Dorrington, Michael G.; Yin, Charles; Tu, Zhongyuan; Pelka, Peter; Whyte, Peter; Guarné, Alba; Sakamoto, Kaori; Bowdish, Dawn M.E.

    2016-01-01

    Macrophage receptor with collagenous structure (MARCO) is a Class A Scavenger Receptor (cA-SR) that recognizes and phagocytoses of a wide variety of pathogens. Most cA-SRs that contain a C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, for the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signalling is less clear. The discovery of a naturally-occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signalling, and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-Like Receptor 2 (TLR2)-mediated pro-inflammatory signalling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, while MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signalling, and MARCO-mediated cellular adhesion. PMID:26888252

  17. Structural analysis of the KRIT1 ankyrin repeat and FERM domains reveals a conformationally stable ARD-FERM interface

    SciTech Connect

    Zhang, Rong; Li, Xiaofeng; Boggon, Titus J.

    2015-10-14

    Cerebral cavernous malformations (CCM) are vascular dysplasias that usually occur in the brain and are associated with mutations in the KRIT1/CCM1, CCM2/MGC4607/OSM/Malcavernin, and PDCD10/CCM3/ TFAR15 genes. Here we report the 2.9 Å crystal structure of the ankyrin repeat domain (ARD) and FERM domain of the protein product of KRIT1 (KRIT1; Krev interaction trapped 1). The crystal structure reveals that the KRIT1 ARD contains 4 ankyrin repeats. There is also an unusual conformation in the ANK4 repeat that is stabilized by Trp-404, and the structure reveals a solvent exposed ankyrin groove. Domain orientations of the three copies within the asymmetric unit suggest a stable interaction between KRIT1 ARD and FERM domains, indicating a globular ARD–FERM module. It resembles the additional F0 domain found N-terminal to the FERM domain of talin. Structural analysis of KRIT1 ARD–FERM highlights surface regions of high evolutionary conservation, and suggests potential sites that could mediate interaction with binding partners. The structure therefore provides a better understanding of KRIT1 at the molecular level.

  18. Structural analysis of the KRIT1 ankyrin repeat and FERM domains reveals a conformationally stable ARD-FERM interface

    PubMed Central

    Zhang, Rong; Li, Xiaofeng; Boggon, Titus J.

    2015-01-01

    Cerebral cavernous malformations (CCM) are vascular dysplasias that usually occur in the brain and are associated with mutations in the KRIT1/CCM1, CCM2/MGC4607/OSM/Malcavernin, and PDCD10/CCM3/TFAR15 genes. Here we report the 2.9 Å crystal structure of the ankyrin repeat domain (ARD) and FERM domain of the protein product of KRIT1 (KRIT1; Krev interaction trapped 1). The crystal structure reveals that the KRIT1 ARD contains 4 ankyrin repeats. There is an unusual conformation in the ANK4 repeat that is stabilized by Trp-404, and the structure reveals a solvent exposed ankyrin groove. Domain orientations of the three copies within the asymmetric unit suggest a stable interaction between KRIT1 ARD and FERM domains, indicating a globular ARD-FERM module. This resembles the additional F0 domain found N-terminal to the FERM domain of talin. Structural analysis of KRIT1 ARD-FERM highlights surface regions of high evolutionary conservation, and suggests potential sites that could mediate interaction with binding partners. The structure therefore provides a better understanding of KRIT1 at the molecular level. PMID:26458359

  19. Structural analysis of the KRIT1 ankyrin repeat and FERM domains reveals a conformationally stable ARD-FERM interface.

    PubMed

    Zhang, Rong; Li, Xiaofeng; Boggon, Titus J

    2015-12-01

    Cerebral cavernous malformations (CCM) are vascular dysplasias that usually occur in the brain and are associated with mutations in the KRIT1/CCM1, CCM2/MGC4607/OSM/Malcavernin, and PDCD10/CCM3/TFAR15 genes. Here we report the 2.9 Å crystal structure of the ankyrin repeat domain (ARD) and FERM domain of the protein product of KRIT1 (KRIT1; Krev interaction trapped 1). The crystal structure reveals that the KRIT1 ARD contains 4 ankyrin repeats. There is an unusual conformation in the ANK4 repeat that is stabilized by Trp-404, and the structure reveals a solvent exposed ankyrin groove. Domain orientations of the three copies within the asymmetric unit suggest a stable interaction between KRIT1 ARD and FERM domains, indicating a globular ARD-FERM module. This resembles the additional F0 domain found N-terminal to the FERM domain of talin. Structural analysis of KRIT1 ARD-FERM highlights surface regions of high evolutionary conservation, and suggests potential sites that could mediate interaction with binding partners. The structure therefore provides a better understanding of KRIT1 at the molecular level.

  20. Crystal Structure of the Mycoplasma arthritidis-Derived Mitogen in Apo Form Reveals a 3D Domain-Swapped Dimer

    SciTech Connect

    Liu, L.; Li, Z; Guo, Y; VanVranken, S; Mourad, W; Li, H

    2010-01-01

    Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular V{beta} elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-{angstrom} resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the 'reconstituted' MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM{sub wt} molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.

  1. Magnetic force microscopy reveals meta-stable magnetic domain states that prevent reliable absolute palaeointensity experiments.

    PubMed

    de Groot, Lennart V; Fabian, Karl; Bakelaar, Iman A; Dekkers, Mark J

    2014-08-22

    Obtaining reliable estimates of the absolute palaeointensity of the Earth's magnetic field is notoriously difficult. The heating of samples in most methods induces magnetic alteration--a process that is still poorly understood, but prevents obtaining correct field values. Here we show induced changes in magnetic domain state directly by imaging the domain configurations of titanomagnetite particles in samples that systematically fail to produce truthful estimates. Magnetic force microscope images were taken before and after a heating step typically used in absolute palaeointensity experiments. For a critical temperature (250 °C), we observe major changes: distinct, blocky domains before heating change into curvier, wavy domains thereafter. These structures appeared unstable over time: after 1-year of storage in a magnetic-field-free environment, the domain states evolved into a viscous remanent magnetization state. Our observations qualitatively explain reported underestimates from otherwise (technically) successful experiments and therefore have major implications for all palaeointensity methods involving heating.

  2. Replication Fork Polarity Gradients Revealed by Megabase-Sized U-Shaped Replication Timing Domains in Human Cell Lines

    PubMed Central

    Baker, Antoine; Audit, Benjamin; Chen, Chun-Long; Moindrot, Benoit; Leleu, Antoine; Guilbaud, Guillaume; Rappailles, Aurélien; Vaillant, Cédric; Goldar, Arach; Mongelard, Fabien; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Thermes, Claude; Arneodo, Alain

    2012-01-01

    In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome. PMID:22496629

  3. Modular dispensability of dysferlin C2 domains reveals rational design for mini-dysferlin molecules.

    PubMed

    Azakir, Bilal A; Di Fulvio, Sabrina; Salomon, Steven; Brockhoff, Marielle; Therrien, Christian; Sinnreich, Michael

    2012-08-10

    Dysferlin is a large transmembrane protein composed of a C-terminal transmembrane domain, two DysF domains, and seven C2 domains that mediate lipid- and protein-binding interactions. Recessive loss-of-function mutations in dysferlin lead to muscular dystrophies, for which no treatment is currently available. The large size of dysferlin precludes its encapsulation into an adeno-associated virus (AAV), the vector of choice for gene delivery to muscle. To design mini-dysferlin molecules suitable for AAV-mediated gene transfer, we tested internally truncated dysferlin constructs, each lacking one of the seven C2 domains, for their ability to localize to the plasma membrane and to repair laser-induced plasmalemmal wounds in dysferlin-deficient human myoblasts. We demonstrate that the dysferlin C2B, C2C, C2D, and C2E domains are dispensable for correct plasmalemmal localization. Furthermore, we show that the C2B, C2C, and C2E domains and, to a lesser extent, the C2D domain are dispensable for dysferlin membrane repair function. On the basis of these results, we designed small dysferlin molecules that can localize to the plasma membrane and reseal laser-induced plasmalemmal injuries and that are small enough to be incorporated into AAV. These results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models.

  4. Modular Dispensability of Dysferlin C2 Domains Reveals Rational Design for Mini-dysferlin Molecules*

    PubMed Central

    Azakir, Bilal A.; Di Fulvio, Sabrina; Salomon, Steven; Brockhoff, Marielle; Therrien, Christian; Sinnreich, Michael

    2012-01-01

    Dysferlin is a large transmembrane protein composed of a C-terminal transmembrane domain, two DysF domains, and seven C2 domains that mediate lipid- and protein-binding interactions. Recessive loss-of-function mutations in dysferlin lead to muscular dystrophies, for which no treatment is currently available. The large size of dysferlin precludes its encapsulation into an adeno-associated virus (AAV), the vector of choice for gene delivery to muscle. To design mini-dysferlin molecules suitable for AAV-mediated gene transfer, we tested internally truncated dysferlin constructs, each lacking one of the seven C2 domains, for their ability to localize to the plasma membrane and to repair laser-induced plasmalemmal wounds in dysferlin-deficient human myoblasts. We demonstrate that the dysferlin C2B, C2C, C2D, and C2E domains are dispensable for correct plasmalemmal localization. Furthermore, we show that the C2B, C2C, and C2E domains and, to a lesser extent, the C2D domain are dispensable for dysferlin membrane repair function. On the basis of these results, we designed small dysferlin molecules that can localize to the plasma membrane and reseal laser-induced plasmalemmal injuries and that are small enough to be incorporated into AAV. These results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models. PMID:22736764

  5. An intelligent inter-domain routing scheme under the consideration of diffserv QoS and energy saving in multi-domain software-defined flexible optical networks

    NASA Astrophysics Data System (ADS)

    Zhao, Jijun; Li, Fengyun; Ren, Danping; Hu, Jinhua; Yao, Qiuyan; Li, Wei

    2016-05-01

    Large scale multi-domain software-defined optical networks (SDON) provisioning has become a key area with increased scalable bandwidth services across wider network regions. Although distributed schemes could achieve lightpath routing by the ergodic process of domain boundary nodes, it increases the complexity of the signaling procedure and deployment of the interface. Moreover, the physical impairments are always the main factor of the infrastructure layer in SDON, which affect the transmission quality of the signal. Meanwhile, with increasing energy consumption of the Internet, it is imperative to design energy-efficient networks. To address the above issues, in this paper, an intelligent inter-domain routing scheme, which is supported by hierarchical control plane architecture, is presented based on sub-topology graph under the consideration of diffserv quality-of-service (QoS) and energy saving. The proposed scheme could achieve multi-domain quality of transmission (QoT), energy aware routing and spectrum assignment (RSA). We explore the scenarios where the multi-domain SDON achieve energy efficiency on the basis of meeting the QoT requirement. The blocking, energy consumption and average set up delay performances of the proposed schemes are estimated. The results indicate that the introduction of sub-topology in multi-domain RSA scheme has the better performance comparing with the distributed scheme.

  6. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains.

    PubMed

    Aten, Jacob A; Stap, Jan; Krawczyk, Przemek M; van Oven, Carel H; Hoebe, Ron A; Essers, Jeroen; Kanaar, Roland

    2004-01-02

    Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.

  7. fMRI reveals reciprocal inhibition between social and physical cognitive domains

    PubMed Central

    Jack, Anthony I.; Dawson, Abigail; Begany, Katelyn; Leckie, Regina L.; Barry, Kevin; Ciccia, Angela; Snyder, Abraham

    2012-01-01

    Two lines of evidence indicate that there exists a reciprocal inhibitory relationship between opposed brain networks. First, most attention-demanding cognitive tasks activate a stereotypical set of brain areas, known as the task-positive network and simultaneously deactivate a different set of brain regions, commonly referred to as the task negative or default mode network. Second, functional connectivity analyses show that these same opposed networks are anti-correlated in the resting state. We hypothesize that these reciprocally inhibitory effects reflect two incompatible cognitive modes, each of which is directed towards understanding the external world. Thus, engaging one mode activates one set of regions and suppresses activity in the other. We test this hypothesis by identifying two types of problem-solving task which, on the basis of prior work, have been consistently associated with the task positive and task negative regions: tasks requiring social cognition, i.e., reasoning about the mental states of other persons, and tasks requiring physical cognition, i.e., reasoning about the causal/mechanical properties of inanimate objects. Social and mechanical reasoning tasks were presented to neurologically normal participants during fMRI. Each task type was presented using both text and video clips. Regardless of presentation modality, we observed clear evidence of reciprocal suppression: social tasks deactivated regions associated with mechanical reasoning and mechanical tasks deactivated regions associated with social reasoning. These findings are not explained by self-referential processes, task engagement, mental simulation, mental time travel or external vs. internal attention, all factors previously hypothesized to explain default mode network activity. Analyses of resting state data revealed a close match between the regions our tasks identified as reciprocally inhibitory and regions of maximal anti-correlation in the resting state. These results indicate

  8. fMRI reveals reciprocal inhibition between social and physical cognitive domains.

    PubMed

    Jack, Anthony I; Dawson, Abigail J; Begany, Katelyn L; Leckie, Regina L; Barry, Kevin P; Ciccia, Angela H; Snyder, Abraham Z

    2013-02-01

    Two lines of evidence indicate that there exists a reciprocal inhibitory relationship between opposed brain networks. First, most attention-demanding cognitive tasks activate a stereotypical set of brain areas, known as the task-positive network and simultaneously deactivate a different set of brain regions, commonly referred to as the task negative or default mode network. Second, functional connectivity analyses show that these same opposed networks are anti-correlated in the resting state. We hypothesize that these reciprocally inhibitory effects reflect two incompatible cognitive modes, each of which may be directed towards understanding the external world. Thus, engaging one mode activates one set of regions and suppresses activity in the other. We test this hypothesis by identifying two types of problem-solving task which, on the basis of prior work, have been consistently associated with the task positive and task negative regions: tasks requiring social cognition, i.e., reasoning about the mental states of other persons, and tasks requiring physical cognition, i.e., reasoning about the causal/mechanical properties of inanimate objects. Social and mechanical reasoning tasks were presented to neurologically normal participants during fMRI. Each task type was presented using both text and video clips. Regardless of presentation modality, we observed clear evidence of reciprocal suppression: social tasks deactivated regions associated with mechanical reasoning and mechanical tasks deactivated regions associated with social reasoning. These findings are not explained by self-referential processes, task engagement, mental simulation, mental time travel or external vs. internal attention, all factors previously hypothesized to explain default mode network activity. Analyses of resting state data revealed a close match between the regions our tasks identified as reciprocally inhibitory and regions of maximal anti-correlation in the resting state. These results

  9. Dissection of the Adenoviral VA RNAI Central Domain Structure Reveals Minimum Requirements for RNA-mediated Inhibition of PKR*

    PubMed Central

    Wilson, Jo L.; Vachon, Virginia K.; Sunita, S.; Schwartz, Samantha L.; Conn, Graeme L.

    2014-01-01

    Virus-associated RNA I (VA RNAI) is a short (∼160-nucleotide) non-coding RNA transcript employed by adenoviruses to subvert the innate immune system protein double-stranded RNA-activated protein kinase (PKR). The central domain of VA RNAI is proposed to contain a complex tertiary structure that contributes to its optimal inhibitory activity against PKR. Here we use a combination of VA RNAI mutagenesis, structural analyses, as well as PKR activity and binding assays to dissect this tertiary structure and assess its functional role. Our results support the existence of a pH- and Mg2+-dependent tertiary structure involving pseudoknot formation within the central domain. Unexpectedly, this structure appears to play no direct role in PKR inhibition. Deletion of central domain sequences within a minimal but fully active construct lacking the tertiary structure reveals a crucial role in PKR binding and inhibition for nucleotides in the 5′ half of the central domain. Deletion of the central domain 3′ half also significantly impacts activity but appears to arise indirectly by reducing its capacity to assist in optimally presenting the 5′ half sequence. Collectively, our results identify regions of VA RNAI critical for PKR inhibition and reveal that the requirements for an effective RNA inhibitor of PKR are simpler than appreciated previously. PMID:24970889

  10. The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3

    SciTech Connect

    Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.

    2007-07-01

    The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

  11. Communication Between RNA Folding Domains Revealed by Folding of Circularly Permuted Ribozymes

    SciTech Connect

    Lease,R.; Adilakshmi, T.; Heilman-Miller, S.; Woodson, S.

    2007-01-01

    To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T1 digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

  12. Communication between RNA folding domains revealed by folding of circularly permuted ribozymes.

    PubMed

    Lease, Richard A; Adilakshmi, Tadepalli; Heilman-Miller, Susan; Woodson, Sarah A

    2007-10-12

    To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 degrees C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 degrees C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T(1) digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

  13. Structures of YAP protein domains reveal promising targets for development of new cancer drugs

    PubMed Central

    Sudol, Marius; Shields, Denis C.; Farooq, Amjad

    2012-01-01

    YAP (Yes-associated protein) is a potent oncogene and a major effector of the mammalian Hippo tumor suppressor pathway. In this review, our emphasis is on the structural basis of how YAP recognizes its various cellular partners. In particular, we discuss the role of LATS kinase and AMOTL1 junction protein, two key cellular partners of YAP that bind to its WW domain, in mediating cytoplasmic localization of YAP and thereby playing a key role in the regulation of its transcriptional activity. Importantly, the crystal structure of an amino-terminal domain of YAP in complex with the carboxy-terminal domain of TEAD transcription factor was only recently solved at atomic resolution, while the structure of WW domain of YAP in complex with a peptide containing the PPxY motif has been available for more than a decade. We discuss how such structural information may be exploited for the rational development of novel anti-cancer therapeutics harboring greater efficacy coupled with low toxicity. We also embark on a brief discussion of how recent in silico studies led to identification of the cardiac glycoside digitoxin as a potential modulator of WW domain-ligand interactions. Conversely, dobutamine was identified in a screen of known drugs as a compound that promotes cytoplasmic localization of YAP, thereby resulting in growth suppressing activity. Finally, we discuss how a recent study on the dynamics of WW domain folding on a biologically critical time scale may provide a tool to generate repertoires of WW domain variants for regulation of the Hippo pathway toward desired, non-oncogenic outputs. PMID:22609812

  14. Structural and Functional Analysis of a Plant Resistance Protein TIR Domain Reveals Interfaces for Self-Association, Signaling, and Autoregulation

    PubMed Central

    Bernoux, Maud; Ve, Thomas; Williams, Simon; Warren, Christopher; Hatters, Danny; Valkov, Eugene; Zhang, Xiaoxiao; Ellis, Jeffrey G.; Kobe, Bostjan; Dodds, Peter N.

    2011-01-01

    SUMMARY The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation. PMID:21402359

  15. Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation.

    PubMed

    Bernoux, Maud; Ve, Thomas; Williams, Simon; Warren, Christopher; Hatters, Danny; Valkov, Eugene; Zhang, Xiaoxiao; Ellis, Jeffrey G; Kobe, Bostjan; Dodds, Peter N

    2011-03-17

    The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

    PubMed Central

    Hyde, Caroline A. C.; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H. Kaspar

    2012-01-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies. PMID:22801374

  17. Functional organization of color domains in V1 and V2 of macaque monkey revealed by optical imaging.

    PubMed

    Lu, Haidong D; Roe, Anna W

    2008-03-01

    Areas V1 and V2 of Macaque monkey visual cortex are characterized by unique cytochrome-oxidase (CO)-staining patterns. Initial electrophysiological studies associated CO blobs in V1 with processing of surface properties such as color and brightness and the interblobs with contour information processing. However, many subsequent studies showed controversial results, some supporting this proposal and others failing to find significant functional differences between blobs and interblobs. In this study, we have used optical imaging to map color-selective responses in V1 and V2. In V1, we find striking "blob-like" patterns of color response. Fine alignment of optical maps and CO-stained tissue revealed that color domains in V1 strongly associate with CO blobs. We also find color domains in V1 align along centers of ocular dominance columns. Furthermore, color blobs in V1 have low orientation selectivity and do not overlap with centers of orientation domains. In V2, color domains coincide with thin stripes; orientation-selective domains coincide with thick and pale stripes. We conclude that color and orientation-selective responses are preferentially located in distinct CO compartments in V1 and V2. We propose that the term "blob" encompasses both the concept of "CO blob" and "color domain" in V1.

  18. Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

    PubMed

    Hyde, Caroline A C; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H Kaspar; Ballmer-Hofer, Kurt

    2012-10-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

  19. Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

    PubMed

    Gu, Yijun; Reshetnikova, Ludmila; Li, Yue; Wu, Yan; Yan, Honggao; Singh, Shivendra; Ji, Xinhua

    2002-06-07

    Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.

  20. High-resolution 2-D Bragg diffraction reveal heterogeneous domain transformation behavior in a bulk relaxor ferroelectric

    SciTech Connect

    Pramanick, Abhijit; Stoica, Alexandru D.; An, Ke

    2016-08-29

    In-situ measurement of fine-structure of neutron Bragg diffraction peaks from a relaxor single-crystal using a time-of-flight instrument reveals highly heterogeneous mesoscale domain transformation behavior under applied electric fields. It is observed that only ∼25% of domains undergo reorientation or phase transition contributing to large average strains, while at least 40% remain invariant and exhibit microstrains. Such insights could be central for designing new relaxor materials with better performance and longevity. The current experimental technique can also be applied to resolve complex mesoscale phenomena in other functional materials.

  1. High-resolution 2-D Bragg diffraction reveal heterogeneous domain transformation behavior in a bulk relaxor ferroelectric

    SciTech Connect

    Pramanick, Abhijit; Stoica, Alexandru D.; An, Ke

    2016-09-02

    In-situ measurement of fine-structure of neutron Bragg diffraction peaks from a relaxor single-crystal using a time-of-flight instrument reveals highly heterogeneous mesoscale domain transformation behavior under applied electric fields. We observed that only 25% of domains undergo reorienta- tion or phase transition contributing to large average strains, while at least 40% remain invariant and exhibit microstrains. Such insights could be central for designing new relaxor materials with better performance and longevity. The current experimental technique can also be applied to resolve com- plex mesoscale phenomena in other functional materials.

  2. High-resolution 2-D Bragg diffraction reveal heterogeneous domain transformation behavior in a bulk relaxor ferroelectric

    DOE PAGES

    Pramanick, Abhijit; Stoica, Alexandru D.; An, Ke

    2016-09-02

    In-situ measurement of fine-structure of neutron Bragg diffraction peaks from a relaxor single-crystal using a time-of-flight instrument reveals highly heterogeneous mesoscale domain transformation behavior under applied electric fields. We observed that only 25% of domains undergo reorienta- tion or phase transition contributing to large average strains, while at least 40% remain invariant and exhibit microstrains. Such insights could be central for designing new relaxor materials with better performance and longevity. The current experimental technique can also be applied to resolve com- plex mesoscale phenomena in other functional materials.

  3. Changepoint detection in base-resolution methylome data reveals a robust signature of methylated domain landscape.

    PubMed

    Yokoyama, Takao; Miura, Fumihito; Araki, Hiromitsu; Okamura, Kohji; Ito, Takashi

    2015-08-12

    Base-resolution methylome data generated by whole-genome bisulfite sequencing (WGBS) is often used to segment the genome into domains with distinct methylation levels. However, most segmentation methods include many parameters to be carefully tuned and/or fail to exploit the unsurpassed resolution of the data. Furthermore, there is no simple method that displays the composition of the domains to grasp global trends in each methylome. We propose to use changepoint detection for domain demarcation based on base-resolution methylome data. While the proposed method segments the methylome in a largely comparable manner to conventional approaches, it has only a single parameter to be tuned. Furthermore, it fully exploits the base-resolution of the data to enable simultaneous detection of methylation changes in even contrasting size ranges, such as focal hypermethylation and global hypomethylation in cancer methylomes. We also propose a simple plot termed methylated domain landscape (MDL) that globally displays the size, the methylation level and the number of the domains thus defined, thereby enabling one to intuitively grasp trends in each methylome. Since the pattern of MDL often reflects cell lineages and is largely unaffected by data size, it can serve as a novel signature of methylome. Changepoint detection in base-resolution methylome data followed by MDL plotting provides a novel method for methylome characterization and will facilitate global comparison among various WGBS data differing in size and even species origin.

  4. Crystal structure of a Ba(2+)-bound gating ring reveals elementary steps in RCK domain activation.

    PubMed

    Smith, Frank J; Pau, Victor P T; Cingolani, Gino; Rothberg, Brad S

    2012-12-05

    RCK domains control activity of a variety of K(+) channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca(2+)-gated K(+) channel, MthK, bound with Ba(2+), at 3.1 Å resolution. The Ba(2+)-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba(2+) bound at several positions. One of the Ba(2+) sites, termed C1, overlaps with a known Ca(2+)-activation site, determined by residues D184 and E210. Functionally, Ba(2+) can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg(2+) (up to 100 mM) was ineffective. Ba(2+) activation was abolished by the mutation D184N, suggesting that Ba(2+) activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture.

    PubMed

    Vietri Rudan, Matteo; Barrington, Christopher; Henderson, Stephen; Ernst, Christina; Odom, Duncan T; Tanay, Amos; Hadjur, Suzana

    2015-03-03

    Topological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C) in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF's long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules.

  6. Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

    PubMed Central

    2015-01-01

    The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W+-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain. PMID:25290210

  7. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  8. The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

    PubMed

    Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C

    2014-10-07

    Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels.

  9. Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs

    PubMed Central

    2010-01-01

    Background As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity. Results In this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present. Conclusions We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity. PMID:20687959

  10. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    SciTech Connect

    S Truschel; D Sengupta; A Foote; A Heroux; M Macbeth; A Linstedt

    2011-12-31

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  11. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    SciTech Connect

    Truschel, S.T.; Heroux, A.; Sengupta, D.; Foote, A.; Macbeth, M. R.; Linstedt, A. D.

    2011-06-10

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  12. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

    PubMed

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

    2017-09-15

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

  13. Spontaneous resting-state BOLD fluctuations reveal persistent domain-specific neural networks

    PubMed Central

    Martin, Alex

    2012-01-01

    Resting-state functional connectivity MRI (rs-fcMRI) analyses have identified intrinsic neural networks supporting domain-general cognitive functions including language, attention, executive control and memory. The brain, however, also has a domain-specific organization, including regions that contribute to perceiving and knowing about others (the ‘social’ system) or manipulable objects designed to perform specific functions (the ‘tool’ system). These ‘social’ and ‘tool’ systems, however, might not constitute intrinsic neural networks per se, but rather only come online as needed to support retrieval of domain-specific information during social- or tool-related cognitive tasks. To address this issue, we functionally localized two regions in lateral temporal cortex activated when subjects perform social- and tool conceptual tasks. We then compared the strength of the correlations with these seed regions during rs-fcMRI. Here, we show that the ‘social’ and ‘tool’ neural networks are maintained even when subjects are not engaged in social- and tool-related information processing, and so constitute intrinsic domain-specific neural networks. PMID:21586527

  14. Mapping of NRAGE domains reveals clues to cell viability in BMP signaling

    PubMed Central

    Rochira, Jennifer A.; Cowling, Rebecca A.; Himmelfarb, Joshua S.; Adams, Tamara L.

    2011-01-01

    Bone morphogenetic signaling (BMP) is a key pathway during neurogenesis and depends on many downstream intermediators to carry out its signaling. One such signaling pathway utilizes neurotrophin receptor-interacting MAGE protein (NRAGE), a member of the melanoma-associated antigen (MAGE) family, to upregulate p38 mitogen activated protein kinase (p38MAPK) in response to cellular stress and activate caspases which are critical in leading cells to death. NRAGE consists of two conserved MAGE homology domains separated by a unique hexapeptide repeat domain. Although we have previously implicated NRAGE in inducing apoptosis in neural progenitors and P19 cells, a model system for neural progenitors, its domains have yet to be explored in determining which one may be responsible for setting up the signaling for apoptosis. Here, we overexpressed a series of deletion mutations in P19 cells to show that only those with at least half of the repeat domain, activated p38MAPK and underwent apoptosis offering intriguing incite into NRAGE’s contribution in BMP apoptotic signaling. PMID:19937275

  15. Mapping of NRAGE domains reveals clues to cell viability in BMP signaling.

    PubMed

    Rochira, Jennifer A; Cowling, Rebecca A; Himmelfarb, Joshua S; Adams, Tamara L; Verdi, Joseph M

    2010-01-01

    Bone morphogenetic signaling (BMP) is a key pathway during neurogenesis and depends on many downstream intermediators to carry out its signaling. One such signaling pathway utilizes neurotrophin receptor-interacting MAGE protein (NRAGE), a member of the melanoma-associated antigen (MAGE) family, to upregulate p38 mitogen activated protein kinase (p38(MAPK)) in response to cellular stress and activate caspases which are critical in leading cells to death. NRAGE consists of two conserved MAGE homology domains separated by a unique hexapeptide repeat domain. Although we have previously implicated NRAGE in inducing apoptosis in neural progenitors and P19 cells, a model system for neural progenitors, its domains have yet to be explored in determining which one may be responsible for setting up the signaling for apoptosis. Here, we overexpressed a series of deletion mutations in P19 cells to show that only those with at least half of the repeat domain, activated p38(MAPK) and underwent apoptosis offering intriguing incite into NRAGE's contribution in BMP apoptotic signaling.

  16. Generic Language Use Reveals Domain Differences in Young Children's Expectations about Animal and Artifact Categories

    ERIC Educational Resources Information Center

    Brandone, Amanda C.; Gelman, Susan A.

    2013-01-01

    The goal of the present study was to explore domain differences in young children's expectations about the structure of animal and artifact categories. We examined 5-year-olds' and adults' use of category-referring generic noun phrases (e.g., "Birds fly") about novel animals and artifacts. The same stimuli served as both animals and artifacts;…

  17. Generic Language Use Reveals Domain Differences in Young Children's Expectations about Animal and Artifact Categories

    ERIC Educational Resources Information Center

    Brandone, Amanda C.; Gelman, Susan A.

    2013-01-01

    The goal of the present study was to explore domain differences in young children's expectations about the structure of animal and artifact categories. We examined 5-year-olds' and adults' use of category-referring generic noun phrases (e.g., "Birds fly") about novel animals and artifacts. The same stimuli served as both animals and artifacts;…

  18. Crystal structure of a beta-finger domain of Prp8 reveals analogy to ribosomal proteins

    SciTech Connect

    Yang, K.; Heroux, A.; Zhang, L.; Zhao, R.

    2008-09-16

    Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a {beta}-hairpin finger protruding out of the protein (hence, this domain will be referred to as the {beta}-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the {beta}-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the {beta}-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its {beta}-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the {beta}-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome.

  19. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms.

    PubMed

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Nicora, Horacio D Lopez; Caetano-Anollés, Gustavo

    2011-11-08

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  20. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    PubMed Central

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

    2011-01-01

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  1. The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain.

    PubMed

    Reinelt, Stefan; Hofmann, Eckhard; Gerharz, Tanja; Bott, Michael; Madden, Dean R

    2003-10-03

    The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.

  2. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    PubMed

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects.

  3. Flexibility and symmetry of prokaryotic genome rearrangement reveal lineage-associated core-gene-defined genome organizational frameworks.

    PubMed

    Kang, Yu; Gu, Chaohao; Yuan, Lina; Wang, Yue; Zhu, Yanmin; Li, Xinna; Luo, Qibin; Xiao, Jingfa; Jiang, Daquan; Qian, Minping; Ahmed Khan, Aftab; Chen, Fei; Zhang, Zhang; Yu, Jun

    2014-11-25

    The prokaryotic pangenome partitions genes into core and dispensable genes. The order of core genes, albeit assumed to be stable under selection in general, is frequently interrupted by horizontal gene transfer and rearrangement, but how a core-gene-defined genome maintains its stability or flexibility remains to be investigated. Based on data from 30 species, including 425 genomes from six phyla, we grouped core genes into syntenic blocks in the context of a pangenome according to their stability across multiple isolates. A subset of the core genes, often species specific and lineage associated, formed a core-gene-defined genome organizational framework (cGOF). Such cGOFs are either single segmental (one-third of the species analyzed) or multisegmental (the rest). Multisegment cGOFs were further classified into symmetric or asymmetric according to segment orientations toward the origin-terminus axis. The cGOFs in Gram-positive species are exclusively symmetric and often reversible in orientation, as opposed to those of the Gram-negative bacteria, which are all asymmetric and irreversible. Meanwhile, all species showing strong strand-biased gene distribution contain symmetric cGOFs and often specific DnaE (α subunit of DNA polymerase III) isoforms. Furthermore, functional evaluations revealed that cGOF genes are hub associated with regard to cellular activities, and the stability of cGOF provides efficient indexes for scaffold orientation as demonstrated by assembling virtual and empirical genome drafts. cGOFs show species specificity, and the symmetry of multisegmental cGOFs is conserved among taxa and constrained by DNA polymerase-centric strand-biased gene distribution. The definition of species-specific cGOFs provides powerful guidance for genome assembly and other structure-based analysis. Prokaryotic genomes are frequently interrupted by horizontal gene transfer (HGT) and rearrangement. To know whether there is a set of genes not only conserved in position

  4. Structure of Human J-type Co-chaperone HscB Reveals a Tetracysteine Metal-binding Domain

    SciTech Connect

    Bitto, Eduard; Bingman, Craig A.; Bittova, Lenka; Kondrashov, Dmitry A.; Bannen, Ryan M.; Fox, Brian G.; Markley, John L.; Phillips, Jr., George N.

    2008-11-24

    Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0 A crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX(9-13)FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals.

  5. NMR Study Reveals the Receiver Domain of Arabidopsis ETHYLENE RESPONSE1 Ethylene Receptor as an Atypical Type Response Regulator

    PubMed Central

    Lee, Yi-Zong; Wen, Chi-Kuang; Sue, Shih-Che

    2016-01-01

    The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation. PMID:27486797

  6. Flexibility and Symmetry of Prokaryotic Genome Rearrangement Reveal Lineage-Associated Core-Gene-Defined Genome Organizational Frameworks

    PubMed Central

    Kang, Yu; Gu, Chaohao; Yuan, Lina; Wang, Yue; Zhu, Yanmin; Li, Xinna; Luo, Qibin; Xiao, Jingfa; Jiang, Daquan; Qian, Minping; Ahmed Khan, Aftab; Chen, Fei

    2014-01-01

    ABSTRACT The prokaryotic pangenome partitions genes into core and dispensable genes. The order of core genes, albeit assumed to be stable under selection in general, is frequently interrupted by horizontal gene transfer and rearrangement, but how a core-gene-defined genome maintains its stability or flexibility remains to be investigated. Based on data from 30 species, including 425 genomes from six phyla, we grouped core genes into syntenic blocks in the context of a pangenome according to their stability across multiple isolates. A subset of the core genes, often species specific and lineage associated, formed a core-gene-defined genome organizational framework (cGOF). Such cGOFs are either single segmental (one-third of the species analyzed) or multisegmental (the rest). Multisegment cGOFs were further classified into symmetric or asymmetric according to segment orientations toward the origin-terminus axis. The cGOFs in Gram-positive species are exclusively symmetric and often reversible in orientation, as opposed to those of the Gram-negative bacteria, which are all asymmetric and irreversible. Meanwhile, all species showing strong strand-biased gene distribution contain symmetric cGOFs and often specific DnaE (α subunit of DNA polymerase III) isoforms. Furthermore, functional evaluations revealed that cGOF genes are hub associated with regard to cellular activities, and the stability of cGOF provides efficient indexes for scaffold orientation as demonstrated by assembling virtual and empirical genome drafts. cGOFs show species specificity, and the symmetry of multisegmental cGOFs is conserved among taxa and constrained by DNA polymerase-centric strand-biased gene distribution. The definition of species-specific cGOFs provides powerful guidance for genome assembly and other structure-based analysis. PMID:25425232

  7. PAS-MEDIATED DIMERIZATION OF SOLUBLE GUANYLYL CYCLASE REVEALED BY SIGNAL TRANSDUCTION HISTIDINE KINASE DOMAIN CRYSTAL STRUCTURE

    PubMed Central

    Ma, Xiaolei; Sayed, Nazish; Baskaran, Padmamalini; Beuve, Annie; van den Akker, Focco

    2010-01-01

    Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) since it is often associated with the heme-nitric-oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 Å resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS-dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCβ1 H-NOXA homodimerization, heterodimerization of full length heterodimeric sGC was mostly unaffected likely due to sGC’s additional dimerization contacts in the coiled-coil and catalytic domains. Exceptions are mutations sGC-α1 F285A and -β1 F217A which each caused a drastic drop in NO stimulated activity and mutations sGCα1 Q368A and -β1 Q309A which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture. PMID:18006497

  8. The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains

    PubMed Central

    Wetherell, Charmaine; Cameron, Caroline E.

    2016-01-01

    Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope. PMID:27832149

  9. Functional Organization of Color Domains in V1 and V2 of Macaque Monkey Revealed by Optical Imaging

    PubMed Central

    Lu, Haidong D.; Roe, Anna W.

    2009-01-01

    Areas V1 and V2 of Macaque monkey visual cortex are characterized by unique cytochrome-oxidase (CO)--staining patterns. Initial electrophysiological studies associated CO blobs in V1 with processing of surface properties such as color and brightness and the interblobs with contour information processing. However, many subsequent studies showed controversial results, some supporting this proposal and others failing to find significant functional differences between blobs and interblobs. In this study, we have used optical imaging to map color-selective responses in V1 and V2. In V1, we find striking “blob-like” patterns of color response. Fine alignment of optical maps and CO-stained tissue revealed that color domains in V1 strongly associate with CO blobs. We also find color domains in V1 align along centers of ocular dominance columns. Furthermore, color blobs in V1 have low orientation selectivity and do not overlap with centers of orientation domains. In V2, color domains coincide with thin stripes; orientation-selective domains coincide with thick and pale stripes. We conclude that color and orientation-selective responses are preferentially located in distinct CO compartments in V1 and V2. We propose that the term “blob” encompasses both the concept of “CO blob” and “color domain” in V1. PMID:17576751

  10. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  11. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    SciTech Connect

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; Pyle, Anna Marie

    2015-10-26

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed and the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.

  12. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    DOE PAGES

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; ...

    2015-10-26

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed andmore » the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.« less

  13. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

    PubMed

    Hopf, Thomas A; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S; Benton, Richard

    2015-01-13

    Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

  14. Analysis of vaccinia virus temperature-sensitive I7L mutants reveals two potential functional domains

    PubMed Central

    Moerdyk, Megan J; Byrd, Chelsea M; Hruby, Dennis E

    2006-01-01

    As an approach to initiating a structure-function analysis of the vaccinia virus I7L core protein proteinase, a collection of conditional-lethal mutants in which the mutation had been mapped to the I7L locus were subjected to genomic sequencing and phenotypic analyses. Mutations in six vaccinia virus I7L temperature sensitive mutants fall into two groups: changes at three positions at the N-terminal end between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic domain. Regardless of the position of the mutation, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested prior to core condensation. Thus it appears that the two clusters of mutations may affect two different functional domains required for proteinase activity. PMID:16945137

  15. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    PubMed Central

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; Pyle, Anna Marie

    2015-01-01

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. Here, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Comprised of intron domain 1 from the Oceanobacillus iheyensis group II intron (D1, 266 nts), this intermediate retains native-like features but adopts a compact conformation in which the active-site cleft is closed. Transition between this closed and open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. The open state is then stabilized by sequential docking of downstream intron domains, suggesting a “first comes, first folds” strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures. PMID:26502156

  16. The nature of domain walls in ultrathin ferromagnets revealed by scanning nanomagnetometry.

    PubMed

    Tetienne, J-P; Hingant, T; Martínez, L J; Rohart, S; Thiaville, A; Diez, L Herrera; Garcia, K; Adam, J-P; Kim, J-V; Roch, J-F; Miron, I M; Gaudin, G; Vila, L; Ocker, B; Ravelosona, D; Jacques, V

    2015-04-01

    The capacity to propagate magnetic domain walls with spin-polarized currents underpins several schemes for information storage and processing using spintronic devices. A key question involves the internal structure of the domain walls, which governs their response to certain current-driven torques such as the spin Hall effect. Here we show that magnetic microscopy based on a single nitrogen-vacancy defect in diamond can provide a direct determination of the internal wall structure in ultrathin ferromagnetic films under ambient conditions. We find pure Bloch walls in Ta/CoFeB(1 nm)/MgO, while left-handed Néel walls are observed in Pt/Co(0.6 nm)/AlOx. The latter indicates the presence of a sizable interfacial Dzyaloshinskii-Moriya interaction, which has strong bearing on the feasibility of exploiting novel chiral states such as skyrmions for information technologies.

  17. Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling.

    PubMed

    Carrara, Marta; Prischi, Filippo; Nowak, Piotr R; Ali, Maruf Mu

    2015-06-03

    Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein-folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X-ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.

  18. Internal Domains of Natural Porous Media Revealed: Critical Locations for Transport, Storage, and Chemical Reaction

    SciTech Connect

    Zachara, John; Brantley, Sue; Chorover, Jon; Ewing, Robert; Kerisit, Sebastien; Liu, Chongxuan; Perfect, Edmund; Rother, Gernot; Stack, Andrew G.

    2016-02-05

    Internal pore domains exist within rocks, lithic fragments, subsurface sediments, and soil aggregates. These domains, termed internal domains in porous media (IDPM), represent a subset of a material’s porosity, contain a significant fraction of their porosity as nanopores, dominate the reactive surface area of diverse media types, and are important locations for chemical reactivity and fluid storage. IDPM are key features controlling hydrocarbon release from shales in hydraulic fracture systems, organic matter decomposition in soil, weathering and soil formation, and contaminant behavior in the vadose zone and groundwater. It is traditionally difficult to interrogate, advances in instrumentation and imaging methods are providing new insights on the physical structures and chemical attributes of IDPM, and their contributions to system behaviors. We discuss analytical methods to characterize IDPM, evaluate information on their size distributions, connectivity, and extended structures; determine whether they exhibit unique chemical reactivity; and assess the potential for their inclusion in reactive transport models. Moreover, ongoing developments in measurement technologies and sensitivity, and computer-assisted interpretation will improve understanding of these critical features in the future. Finally, impactful research opportunities exist to advance understanding of IDPM, and to incorporate their effects in reactive transport models for improved environmental simulation and prediction.

  19. Internal Domains of Natural Porous Media Revealed: Critical Locations for Transport, Storage, and Chemical Reaction

    DOE PAGES

    Zachara, John; Brantley, Sue; Chorover, Jon; ...

    2016-02-05

    Internal pore domains exist within rocks, lithic fragments, subsurface sediments, and soil aggregates. These domains, termed internal domains in porous media (IDPM), represent a subset of a material’s porosity, contain a significant fraction of their porosity as nanopores, dominate the reactive surface area of diverse media types, and are important locations for chemical reactivity and fluid storage. IDPM are key features controlling hydrocarbon release from shales in hydraulic fracture systems, organic matter decomposition in soil, weathering and soil formation, and contaminant behavior in the vadose zone and groundwater. It is traditionally difficult to interrogate, advances in instrumentation and imaging methodsmore » are providing new insights on the physical structures and chemical attributes of IDPM, and their contributions to system behaviors. We discuss analytical methods to characterize IDPM, evaluate information on their size distributions, connectivity, and extended structures; determine whether they exhibit unique chemical reactivity; and assess the potential for their inclusion in reactive transport models. Moreover, ongoing developments in measurement technologies and sensitivity, and computer-assisted interpretation will improve understanding of these critical features in the future. Finally, impactful research opportunities exist to advance understanding of IDPM, and to incorporate their effects in reactive transport models for improved environmental simulation and prediction.« less

  20. Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization

    PubMed Central

    Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

    2016-01-01

    Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation. PMID:27811944

  1. Internal Domains of Natural Porous Media Revealed: Critical Locations for Transport, Storage, and Chemical Reaction.

    PubMed

    Zachara, John; Brantley, Sue; Chorover, Jon; Ewing, Robert; Kerisit, Sebastien; Liu, Chongxuan; Perfect, Edmund; Rother, Gernot; Stack, Andrew G

    2016-03-15

    Internal pore domains exist within rocks, lithic fragments, subsurface sediments, and soil aggregates. These domains, termed internal domains in porous media (IDPM), represent a subset of a material's porosity, contain a significant fraction of their porosity as nanopores, dominate the reactive surface area of diverse media types, and are important locations for chemical reactivity and fluid storage. IDPM are key features controlling hydrocarbon release from shales in hydraulic fracture systems, organic matter decomposition in soil, weathering and soil formation, and contaminant behavior in the vadose zone and groundwater. Traditionally difficult to interrogate, advances in instrumentation and imaging methods are providing new insights on the physical structures and chemical attributes of IDPM, and their contributions to system behaviors. Here we discuss analytical methods to characterize IDPM, evaluate information on their size distributions, connectivity, and extended structures; determine whether they exhibit unique chemical reactivity; and assess the potential for their inclusion in reactive transport models. Ongoing developments in measurement technologies and sensitivity, and computer-assisted interpretation will improve understanding of these critical features in the future. Impactful research opportunities exist to advance understanding of IDPM, and to incorporate their effects in reactive transport models for improved environmental simulation and prediction.

  2. Interaction with Polyglutamine Aggregates Reveals a Q/N-rich Domain in TDP-43*

    PubMed Central

    Fuentealba, Rodrigo A.; Udan, Maria; Bell, Shaughn; Wegorzewska, Iga; Shao, Jieya; Diamond, Marc I.; Weihl, Conrad C.; Baloh, Robert H.

    2010-01-01

    The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases. PMID:20554523

  3. Interaction with polyglutamine aggregates reveals a Q/N-rich domain in TDP-43.

    PubMed

    Fuentealba, Rodrigo A; Udan, Maria; Bell, Shaughn; Wegorzewska, Iga; Shao, Jieya; Diamond, Marc I; Weihl, Conrad C; Baloh, Robert H

    2010-08-20

    The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases.

  4. Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis.

    PubMed Central

    Lupas, A; Engelhardt, H; Peters, J; Santarius, U; Volker, S; Baumeister, W

    1994-01-01

    The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacillus sphaericus and Thermus thermophilus, Omp alpha from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins. Images PMID:8113161

  5. Influence of semisynthetic modification of the scaffold of a contact domain of HbS on polymerization: role of flexible surface topology in polymerization inhibition.

    PubMed

    Sonati, Srinivasulu; Bhutoria, Savita; Prabhakaran, Muthuchidambaran; Acharya, Seetharama A

    2017-03-01

    A new variant of HbS, HbS-Einstein with a deletion of segment α23-26 in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain. The design of HbS-Einstein has been undertaken to map the influence of perturbation of molecular surface topology and the flexibility of surface residues in the polymerization. The internal deletion exerts a strong inhibitory influence on Val-6 (β)-dependent polymerization, comparable to single contact site mutations and not for complete neutralization of Val-6(β)-dependent polymerization. The scaffold modification in cis-dimer is inhibitory, and is without any effect when present on the trans dimer. The flexibility changes in the surface topology in the region of scaffold modification apparently counteracts the intrinsic polymerization potential of the molecule. The inhibition is close to that of Le Lamentin mutation [His-20 (α) → Gln] wherein a mutation engineered without much change in flexibility of the contact domain. Interestingly, the chimeric HbS with swine-human chimeric α chain with multiple non-conservative mutations completely inhibits the Val-6(β)-dependent polymerization. The deformabilities of surface topology of chimeric HbS are comparable to HbS in spite of the multiple contact site mutations in the α-chain. We conclude that the design of antisickling Hbs for gene therapy of sickle cell disease should involve multiple mutations of intermolecular contact sites.

  6. Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate

    PubMed Central

    Das, Debasis; Kuzmic, Petr

    2017-01-01

    Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus. Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs. PMID:28630348

  7. Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate.

    PubMed

    Das, Debasis; Kuzmic, Petr; Imperiali, Barbara

    2017-07-03

    Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs.

  8. Internal Domains of Natural Porous Media Revealed: Critical Locations for Transport, Storage, and Chemical Reaction

    SciTech Connect

    Zachara, John M.; Brantley, Susan L.; Chorover, Jon D.; Ewing, Robert P.; Kerisit, Sebastien N.; Liu, Chongxuan; Perfect, E.; Rother, Gernot; Stack, Andrew G.

    2016-03-16

    Internal pore domains exist within rocks, lithic fragments, subsurface sediments and soil aggregates. These domains, which we term internal domains in porous media (IDPM), contain a significant fraction of their porosity as nanopores, dominate the reactive surface area of diverse porous media types, and are important locations for chemical reactivity and hydrocarbon storage. Traditionally difficult to interrogate, advances in instrumentation and imaging methods are providing new insights on the physical structures and chemical attributes of IDPM. In this review we: discuss analytical methods to characterize IDPM, evaluate what has been learned about their size distributions, connectivity, and extended structures; determine whether they exhibit unique chemical reactivity; and assess potential for their inclusion in reactive transport models. Three key findings are noteworthy. 1) A combination of methods now allows complete characterization of the porosity spectrum of natural materials and its connectivity; while imaging microscopies are providing three dimensional representations of the interconnected pore network. 2) Chemical reactivity in pores <10 nm is expected to be different from micro and macropores, yet research performed to date is inconclusive on the nature, direction, and magnitude of effect. 3) Existing continuum reactive transport models treat IDPM as a sub-grid feature with average, empirical, scale-dependent parameters; and are not formulated to include detailed information on pore networks. Overall we find that IDPM are key features controlling hydrocarbon release from shales in hydrofracking systems, organic matter stabilization and recalcitrance in soil, weathering and soil formation, and long term inorganic and organic contaminant behavior in the vadose zone and groundwater. We conclude with an assessment of impactful research opportunities to advance understanding of IDPM, and to incorporate their important effects in reactive transport models

  9. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.

    PubMed

    Sekereš, Juraj; Pejchar, Přemysl; Šantrůček, Jiří; Vukašinović, Nemanja; Žárský, Viktor; Potocký, Martin

    2017-03-01

    The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell. © 2017 American Society of Plant Biologists. All Rights Reserved.

  10. The elusive activity of the Yersinia protein kinase A kinase domain is revealed.

    PubMed

    Laskowski-Arce, Michelle A; Orth, Kim

    2007-10-01

    Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.

  11. Fra Angelico's painting technique revealed by terahertz time-domain imaging (THz-TDI)

    NASA Astrophysics Data System (ADS)

    Koch Dandolo, Corinna Ludovica; Picollo, Marcello; Cucci, Costanza; Jepsen, Peter Uhd

    2016-10-01

    We have investigated with terahertz time-domain imaging (THz-TDI) the well-known Lamentation over the dead Christ panel painting (San Marco Museum, Florence) painted by Fra Giovanni Angelico within 1436 and 1441. The investigation provided a better understanding of the construction and gilding technique used by the eminent artist, as well as the plastering technique used during the nineteenth-century restoration intervention. The evidence obtained from THz-TDI scans was correlated with the available documentation on the preservation history of the art piece. Erosion and damages documented for the wooden support, especially in the lower margin, found confirmation in the THz-TD images.

  12. A numerical approach for simulating fluid structure interaction of flexible thin shells undergoing arbitrarily large deformations in complex domains

    NASA Astrophysics Data System (ADS)

    Gilmanov, Anvar; Le, Trung Bao; Sotiropoulos, Fotis

    2015-11-01

    We present a new numerical methodology for simulating fluid-structure interaction (FSI) problems involving thin flexible bodies in an incompressible fluid. The FSI algorithm uses the Dirichlet-Neumann partitioning technique. The curvilinear immersed boundary method (CURVIB) is coupled with a rotation-free finite element (FE) model for thin shells enabling the efficient simulation of FSI problems with arbitrarily large deformation. Turbulent flow problems are handled using large-eddy simulation with the dynamic Smagorinsky model in conjunction with a wall model to reconstruct boundary conditions near immersed boundaries. The CURVIB and FE solvers are coupled together on the flexible solid-fluid interfaces where the structural nodal positions, displacements, velocities and loads are calculated and exchanged between the two solvers. Loose and strong coupling FSI schemes are employed enhanced by the Aitken acceleration technique to ensure robust coupling and fast convergence especially for low mass ratio problems. The coupled CURVIB-FE-FSI method is validated by applying it to simulate two FSI problems involving thin flexible structures: 1) vortex-induced vibrations of a cantilever mounted in the wake of a square cylinder at different mass ratios and at low Reynolds number; and 2) the more challenging high Reynolds number problem involving the oscillation of an inverted elastic flag. For both cases the computed results are in excellent agreement with previous numerical simulations and/or experiential measurements. Grid convergence tests/studies are carried out for both the cantilever and inverted flag problems, which show that the CURVIB-FE-FSI method provides their convergence. Finally, the capability of the new methodology in simulations of complex cardiovascular flows is demonstrated by applying it to simulate the FSI of a tri-leaflet, prosthetic heart valve in an anatomic aorta and under physiologic pulsatile conditions.

  13. A numerical approach for simulating fluid structure interaction of flexible thin shells undergoing arbitrarily large deformations in complex domains

    SciTech Connect

    Gilmanov, Anvar; Le, Trung Bao; Sotiropoulos, Fotis

    2015-11-01

    We present a new numerical methodology for simulating fluid–structure interaction (FSI) problems involving thin flexible bodies in an incompressible fluid. The FSI algorithm uses the Dirichlet–Neumann partitioning technique. The curvilinear immersed boundary method (CURVIB) is coupled with a rotation-free finite element (FE) model for thin shells enabling the efficient simulation of FSI problems with arbitrarily large deformation. Turbulent flow problems are handled using large-eddy simulation with the dynamic Smagorinsky model in conjunction with a wall model to reconstruct boundary conditions near immersed boundaries. The CURVIB and FE solvers are coupled together on the flexible solid–fluid interfaces where the structural nodal positions, displacements, velocities and loads are calculated and exchanged between the two solvers. Loose and strong coupling FSI schemes are employed enhanced by the Aitken acceleration technique to ensure robust coupling and fast convergence especially for low mass ratio problems. The coupled CURVIB-FE-FSI method is validated by applying it to simulate two FSI problems involving thin flexible structures: 1) vortex-induced vibrations of a cantilever mounted in the wake of a square cylinder at different mass ratios and at low Reynolds number; and 2) the more challenging high Reynolds number problem involving the oscillation of an inverted elastic flag. For both cases the computed results are in excellent agreement with previous numerical simulations and/or experiential measurements. Grid convergence tests/studies are carried out for both the cantilever and inverted flag problems, which show that the CURVIB-FE-FSI method provides their convergence. Finally, the capability of the new methodology in simulations of complex cardiovascular flows is demonstrated by applying it to simulate the FSI of a tri-leaflet, prosthetic heart valve in an anatomic aorta and under physiologic pulsatile conditions.

  14. Single-cell sequencing of Thiomargarita reveals genomic flexibility for adaptation to dynamic redox conditions

    SciTech Connect

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-06-21

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiornargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In conclusion, the

  15. In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

    PubMed Central

    Geslain, R; Martin, F; Delagoutte, B; Cavarelli, J; Gangloff, J; Eriani, G

    2000-01-01

    Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID:10744027

  16. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping.

    PubMed

    Roh, Tae-Young; Cuddapah, Suresh; Zhao, Keji

    2005-03-01

    The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study are significantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements.

  17. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping

    PubMed Central

    Roh, Tae-Young; Cuddapah, Suresh; Zhao, Keji

    2005-01-01

    The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study are significantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements. PMID:15706033

  18. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    PubMed Central

    Sükösd, Zsuzsanna; Andersen, Ebbe S.; Seemann, Stefan E.; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-01-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  19. Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain.

    PubMed

    Sami, Furqan; Gary, Ronald K; Fang, Yayin; Sharma, Sudha

    2016-08-01

    RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQ1 contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.

  20. Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers

    PubMed Central

    Arumughan, Anup; Roske, Yvette; Barth, Carolin; Forero, Laura Lleras; Bravo-Rodriguez, Kenny; Redel, Alexandra; Kostova, Simona; McShane, Erik; Opitz, Robert; Faelber, Katja; Rau, Kirstin; Mielke, Thorsten; Daumke, Oliver; Selbach, Matthias; Sanchez-Garcia, Elsa; Rocks, Oliver; Panáková, Daniela; Heinemann, Udo; Wanker, Erich E.

    2016-01-01

    Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity. PMID:27762274

  1. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-02

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus.

  2. Function of the ATR N-terminal domain revealed by an ATM/ATR chimera

    SciTech Connect

    Chen Xinping; Zhao Runxiang; Glick, Gloria G.; Cortez, David . E-mail: david.cortez@vanderbilt.edu

    2007-05-01

    The ATM and ATR kinases function at the apex of checkpoint signaling pathways. These kinases share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping roles in initiating cell cycle checkpoints. However, they sense DNA damage through distinct mechanisms. ATR primarily senses single stranded DNA (ssDNA) through its interaction with ATRIP, and ATM senses double strand breaks through its interaction with Nbs1. We determined that the N-terminus of ATR contains a domain that binds ATRIP. Attaching this domain to ATM allowed the fusion protein (ATM*) to bind ATRIP and associate with RPA-coated ssDNA. ATM* also gained the ability to localize efficiently to stalled replication forks as well as double strand breaks. Despite having normal kinase activity when tested in vitro and being phosphorylated on S1981 in vivo, ATM* is defective in checkpoint signaling and does not complement cellular deficiencies in either ATM or ATR. These data indicate that the N-terminus of ATR is sufficient to bind ATRIP and to promote localization to sites of replication stress.

  3. Deletion analysis of AGD1 reveals domains crucial for plasma membrane recruitment and function in root hair polarity.

    PubMed

    Yoo, Cheol-Min; Naramoto, Satoshi; Sparks, J Alan; Khan, Bibi Rafeiza; Nakashima, Jin; Fukuda, Hiroo; Blancaflor, Elison B

    2017-06-23

    AGD1, a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP), functions in specifying root hair polarity in Arabidopsis thaliana To better understand how AGD1 modulates root hair growth, we generated full-length and domain-deleted AGD1-green fluorescent protein (GFP) constructs, and followed their localization during root hair development. AGD1-GFP localized to the cytoplasm and was recruited to specific regions of the root hair plasma membrane (PM). Distinct PM AGD1-GFP signal was first detected along the site of root hair bulge formation. The construct continued to mark the PM at the root hair apical dome, but only during periods of reduced growth. During rapid tip growth, AGD1-GFP labeled the PM of the lateral flanks and dissipated from the apical-most PM. Deletion analysis and a single domain GFP fusion revealed that the pleckstrin homology (PH) domain is the minimal unit required for recruitment of AGD1 to the PM. Our results indicate that differential recruitment of AGD1 to specific PM domains is an essential component of the membrane trafficking machinery that facilitates root hair developmental phase transitions and responses to changes in the root microenvironment. © 2017. Published by The Company of Biologists Ltd.

  4. Structure of Human Synaptotagmin 1 C2AB in the Absence of Ca**2+ Reveals a Novel Domain Association

    SciTech Connect

    Fuson, K.L.; Montes, M.; Robert, J.J.; Sutton, R.B.

    2009-06-01

    Release of neurotransmitter from synaptic vesicles requires the Ca{sup 2+}/phospholipid-binding protein synaptotagmin 1. There is considerable evidence that cooperation between the tandem C2 domains of synaptotagmin is a requirement of regulated exocytosis; however, high-resolution structural evidence for this interaction has been lacking. The 2.7 A crystal structure of the cytosolic domains of human synaptotagmin 1 in the absence of Ca{sup 2+} reveals a novel closed conformation of the protein. The shared interface between C2A and C2B is stabilized by a network of interactions between residues on the C-terminal alpha-helix of the C2B domain and residues on loops 1-3 of the Ca{sup 2+}-binding region of C2A. These interactions alter the overall shape of the Ca{sup 2+}-binding pocket of C2A, but not that of C2B. Thus, synaptotagmin 1 C2A-C2B may utilize a novel regulatory mechanism whereby one C2 domain could regulate the other until an appropriate triggering event decouples them.

  5. Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.

    PubMed

    Czabotar, Peter E; Westphal, Dana; Dewson, Grant; Ma, Stephen; Hockings, Colin; Fairlie, W Douglas; Lee, Erinna F; Yao, Shenggen; Robin, Adeline Y; Smith, Brian J; Huang, David C S; Kluck, Ruth M; Adams, Jerry M; Colman, Peter M

    2013-01-31

    In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

  6. Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex

    PubMed Central

    Berke, Ian C.; Boehmer, Thomas; Blobel, Günter; Schwartz, Thomas U.

    2004-01-01

    Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only ∼30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed β-propeller. The surface properties and conservation of the Nup133 β-propeller suggest it may mediate multiple interactions with other proteins. Other β-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex. PMID:15557116

  7. Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria.

    PubMed

    Chadha, Pooja; Sarfo, Akua; Zhang, Dan; Abraham, Thomas; Carmichael, Jillian; Han, Jun; Wills, John W

    2017-01-15

    The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined.

  8. A Flexible Domain-Domain Hinge Promotes an Induced-fit Dominant Mechanism for the Loading of Guide-DNA into Argonaute Protein in Thermus thermophilus.

    PubMed

    Zhu, Lizhe; Jiang, Hanlun; Sheong, Fu Kit; Cui, Xuefeng; Gao, Xin; Wang, Yanli; Huang, Xuhui

    2016-03-17

    Argonaute proteins (Ago) are core components of the RNA Induced Silencing Complex (RISC) that load and utilize small guide nucleic acids to silence mRNAs or cleave foreign DNAs. Despite the essential role of Ago in gene regulation and defense against virus, the molecular mechanism of guide-strand loading into Ago remains unclear. We explore such a mechanism in the bacterium Thermus thermophilus Ago (TtAgo), via a computational approach combining molecular dynamics, bias-exchange metadynamics, and protein-DNA docking. We show that apo TtAgo adopts multiple closed states that are unable to accommodate guide-DNA. Conformations able to accommodate the guide are beyond the reach of thermal fluctuations from the closed states. These results suggest an induced-fit dominant mechanism for guide-strand loading in TtAgo, drastically different from the two-step mechanism for human Ago 2 (hAgo2) identified in our previous study. Such a difference between TtAgo and hAgo2 is found to mainly originate from the distinct rigidity of their L1-PAZ hinge. Further comparison among known Ago structures from various species indicates that the L1-PAZ hinge may be flexible in general for prokaryotic Ago's but rigid for eukaryotic Ago's.

  9. A previously unobserved conformation for the human Pex5p receptor suggests roles for intrinsic flexibility and rigid domain motions in ligand binding

    PubMed Central

    Stanley, Will A; Pursiainen, Niko V; Garman, Elspeth F; Juffer, André H; Wilmanns, Matthias; Kursula, Petri

    2007-01-01

    Background The C-terminal tetratricopeptide (TPR) repeat domain of Pex5p recognises proteins carrying a peroxisomal targeting signal type 1 (PTS1) tripeptide in their C-terminus. Previously, structural data have been obtained from the TPR domain of Pex5p in both the liganded and unliganded states, indicating a conformational change taking place upon cargo protein binding. Such a conformational change would be expected to play a major role both during PTS1 protein recognition as well as in cargo release into the peroxisomal lumen. However, little information is available on the factors that may regulate such structural changes. Results We have used a range of biophysical and computational methods to further analyse the conformational flexibility and ligand binding of Pex5p. A new crystal form for the human Pex5p C-terminal domain (Pex5p(C)) was obtained in the presence of Sr2+ ions, and the structure presents a novel conformation, distinct from all previous liganded and apo crystal structures for Pex5p(C). The difference relates to a near-rigid body movement of two halves of the molecule, and this movement is different from that required to reach a ring-like conformation upon PTS1 ligand binding. The bound Sr2+ ion changes the dynamic properties of Pex5p(C) affecting its conformation, possibly by making the Sr2+-binding loop – located near the hinge region for the observed domain motions – more rigid. Conclusion The current data indicate that Pex5p(C) is able to sample a range of conformational states in the absence of bound PTS1 ligand. The domain movements between various apo conformations are distinct from those involved in ligand binding, although the differences between all observed conformations so far can be characterised by the movement of the two halves of Pex5p(C) as near-rigid bodies with respect to each other. PMID:17428317

  10. Touchscreen-paradigm for mice reveals cross-species evidence for an antagonistic relationship of cognitive flexibility and stability

    PubMed Central

    Richter, S. Helene; Vogel, Anne S.; Ueltzhöffer, Kai; Muzzillo, Chiara; Vogt, Miriam A.; Lankisch, Katja; Armbruster-Genç, Diana J. N.; Riva, Marco A.; Fiebach, Christian J.; Gass, Peter; Vollmayr, Barbara

    2014-01-01

    The abilities to either flexibly adjust behavior according to changing demands (cognitive flexibility) or to maintain it in the face of potential distractors (cognitive stability) are critical for adaptive behavior in many situations. Recently, a novel human paradigm has found individual differences of cognitive flexibility and stability to be related to common prefrontal networks. The aims of the present study were, first, to translate this paradigm from humans to mice and, second, to test conceptual predictions of a computational model of prefrontal working memory mechanisms, the Dual State Theory, which assumes an antagonistic relation between cognitive flexibility and stability. Mice were trained in a touchscreen-paradigm to discriminate visual cues. The task involved “ongoing” and cued “switch” trials. In addition distractor cues were interspersed to test the ability to resist distraction, and an ambiguous condition assessed the spontaneous switching between two possible responses without explicit cues. While response times did not differ substantially between conditions, error rates (ER) increased from the “ongoing” baseline condition to the most complex condition, where subjects were required to switch between two responses in the presence of a distracting cue. Importantly, subjects switching more often spontaneously were found to be more distractible by task irrelevant cues, but also more flexible in situations, where switching was required. These results support a dichotomy of cognitive flexibility and stability as predicted by the Dual State Theory. Furthermore, they replicate critical aspects of the human paradigm, which indicates the translational potential of the testing procedure and supports the use of touchscreen procedures in preclinical animal research. PMID:24834036

  11. Touchscreen-paradigm for mice reveals cross-species evidence for an antagonistic relationship of cognitive flexibility and stability.

    PubMed

    Richter, S Helene; Vogel, Anne S; Ueltzhöffer, Kai; Muzzillo, Chiara; Vogt, Miriam A; Lankisch, Katja; Armbruster-Genç, Diana J N; Riva, Marco A; Fiebach, Christian J; Gass, Peter; Vollmayr, Barbara

    2014-01-01

    The abilities to either flexibly adjust behavior according to changing demands (cognitive flexibility) or to maintain it in the face of potential distractors (cognitive stability) are critical for adaptive behavior in many situations. Recently, a novel human paradigm has found individual differences of cognitive flexibility and stability to be related to common prefrontal networks. The aims of the present study were, first, to translate this paradigm from humans to mice and, second, to test conceptual predictions of a computational model of prefrontal working memory mechanisms, the Dual State Theory, which assumes an antagonistic relation between cognitive flexibility and stability. Mice were trained in a touchscreen-paradigm to discriminate visual cues. The task involved "ongoing" and cued "switch" trials. In addition distractor cues were interspersed to test the ability to resist distraction, and an ambiguous condition assessed the spontaneous switching between two possible responses without explicit cues. While response times did not differ substantially between conditions, error rates (ER) increased from the "ongoing" baseline condition to the most complex condition, where subjects were required to switch between two responses in the presence of a distracting cue. Importantly, subjects switching more often spontaneously were found to be more distractible by task irrelevant cues, but also more flexible in situations, where switching was required. These results support a dichotomy of cognitive flexibility and stability as predicted by the Dual State Theory. Furthermore, they replicate critical aspects of the human paradigm, which indicates the translational potential of the testing procedure and supports the use of touchscreen procedures in preclinical animal research.

  12. Mechanical implications of the domain structure of fiber-forming collagens: comparison of the molecular and fibrillar flexibilities of the alpha1-chains found in types I-III collagen.

    PubMed

    Silver, Frederick H; Horvath, Istvan; Foran, David J

    2002-05-21

    Fibrillar collagens store, transmit and dissipate elastic energy during tensile deformation. Results of previous studies suggest that the collagen molecule is made up of alternating rigid and flexible domains, and extension of the flexible domains is associated with elastic energy storage. In this study, we model the flexibility of the alpha1-chains found in types I-III collagen molecules and microfibrils in order to understand the molecular basis of elastic energy storage in collagen fibers by analysing the areas under conformational plots for dipeptide sequences. Results of stereochemical modeling suggest that the collagen triple helix is made up of rigid and flexible domains that alternate with periods that are multiples of three amino acid residues. The relative flexibility of dipeptide sequences found in the flexible regions is about a factor of five higher than that found for the flexibility of the rigid regions, and the flexibility of types II and III collagen molecules appears to be higher than that found for the type I collagen molecule. The different collagen alpha1-chains were compared by correlating the flexibilities. The results suggest that the flexibilities of the alpha1-chains of types I and III collagen are more closely related than the flexibilities of the alpha1-chains in types I and II and II and III collagen. The flexible domains found in the alpha1-chains of types I-III collagen were found to be conserved in the microfibril and had periods of about 15 amino acid residues and multiples thereof. The flexibility profiles of types I and II collagen microfibrils were found to be more highly correlated than those for types I and III and II and III. These results suggest that the domain structure of the alpha1-chains found in types I-III collagen is an efficient means for storage of elastic energy during stretching while preserving the triple helical structure of the overall molecule. It is proposed that all collagens that form fibers are designed to

  13. The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing

    SciTech Connect

    Jackson, R.N.; Robinson, H.; Klauer, A. A.; Hintze, B. J.; van Hoof, A.; Johnson, S. J.

    2010-07-01

    The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.

  14. Edge morphology evolution of graphene domains during chemical vapor deposition cooling revealed through hydrogen etching.

    PubMed

    Zhang, Haoran; Zhang, Yanhui; Zhang, Yaqian; Chen, Zhiying; Sui, Yanping; Ge, Xiaoming; Yu, Guanghui; Jin, Zhi; Liu, Xinyu

    2016-02-21

    During cooling, considerable changes such as wrinkle formation and edge passivation occur in graphene synthesized on the Cu substrate. Wrinkle formation is caused by the difference in the thermal expansion coefficients of graphene and its substrate. This work emphasizes the cooling-induced edge passivation. The graphene-edge passivation can limit the regrowth of graphene at the domain edge. Our work shows that silicon-containing particles tend to accumulate at the graphene edge, and the formation of these particles is related to cooling. Furthermore, a clear curvature can be observed at the graphene edge on the Cu substrate, indicating the sinking of the graphene edge into the Cu substrate. Both the sinking of the graphene edge and the accumulation of silicon-containing particles are responsible for edge passivation. In addition, two kinds of graphene edge morphologies are observed after etching, which were explained by different etching mechanisms that illustrate the changes of the graphene edge during cooling.

  15. The structure of Zika virus NS5 reveals a conserved domain conformation.

    PubMed

    Wang, Boxiao; Tan, Xiao-Feng; Thurmond, Stephanie; Zhang, Zhi-Min; Lin, Asher; Hai, Rong; Song, Jikui

    2017-03-27

    The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. The activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV.

  16. The structure of Zika virus NS5 reveals a conserved domain conformation

    DOE PAGES

    Wang, Boxiao; Tan, Xiao -Feng; Thurmond, Stephanie; ...

    2017-03-27

    The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. In conclusion, the activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antiviralsmore » for ZIKV.« less

  17. The structure of Zika virus NS5 reveals a conserved domain conformation

    PubMed Central

    Wang, Boxiao; Tan, Xiao-Feng; Thurmond, Stephanie; Zhang, Zhi-Min; Lin, Asher; Hai, Rong; Song, Jikui

    2017-01-01

    The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. The activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV. PMID:28345600

  18. Image-Based Modeling Reveals Dynamic Redistribution of DNA Damageinto Nuclear Sub-Domains

    SciTech Connect

    Costes Sylvain V., Ponomarev Artem, Chen James L.; Nguyen, David; Cucinotta, Francis A.; Barcellos-Hoff, Mary Helen

    2007-08-03

    Several proteins involved in the response to DNA doublestrand breaks (DSB) f orm microscopically visible nuclear domains, orfoci, after exposure to ionizing radiation. Radiation-induced foci (RIF)are believed to be located where DNA damage occurs. To test thisassumption, we analyzed the spatial distribution of 53BP1, phosphorylatedATM, and gammaH2AX RIF in cells irradiated with high linear energytransfer (LET) radiation and low LET. Since energy is randomly depositedalong high-LET particle paths, RIF along these paths should also berandomly distributed. The probability to induce DSB can be derived fromDNA fragment data measured experimentally by pulsed-field gelelectrophoresis. We used this probability in Monte Carlo simulations topredict DSB locations in synthetic nuclei geometrically described by acomplete set of human chromosomes, taking into account microscope opticsfrom real experiments. As expected, simulations produced DNA-weightedrandom (Poisson) distributions. In contrast, the distributions of RIFobtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) werenon-random. This deviation from the expected DNA-weighted random patterncan be further characterized by "relative DNA image measurements." Thisnovel imaging approach shows that RIF were located preferentially at theinterface between high and low DNA density regions, and were morefrequent than predicted in regions with lower DNA density. The samepreferential nuclear location was also measured for RIF induced by 1 Gyof low-LET radiation. This deviation from random behavior was evidentonly 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AXand 53BP1 RIF showed pronounced deviations up to 30 min after exposure.These data suggest that DNA damage induced foci are restricted to certainregions of the nucleus of human epithelial cells. It is possible that DNAlesions are collected in these nuclear sub-domains for more efficientrepair.

  19. Mutations in the parainfluenza virus 5 fusion protein reveal domains important for fusion triggering and metastability.

    PubMed

    Bose, Sayantan; Heath, Carissa M; Shah, Priya A; Alayyoubi, Maher; Jardetzky, Theodore S; Lamb, Robert A

    2013-12-01

    Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.

  20. Chromosome Model reveals Dynamic Redistribution of DNA Damage into Nuclear Sub-domains

    NASA Technical Reports Server (NTRS)

    Costes, Sylvain V.; Ponomarev, Artem; Chen, James L.; Cucinotta, Francis A.; Barcellos-Hoff, Helen

    2007-01-01

    Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage is induced. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM and gammaH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by relative DNA image measurements. This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent in regions with lower density DNA than predicted. This deviation from random behavior was more pronounced within the first 5 min following irradiation for phosphorylated ATM RIF, while gammaH2AX and 53BP1 RIF showed very pronounced deviation up to 30 min after exposure. These data suggest the existence of repair centers in mammalian epithelial cells. These centers would be nuclear sub-domains where DNA lesions would be collected for more efficient repair.

  1. Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

    PubMed Central

    Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.

    2013-01-01

    Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger. PMID:24089572

  2. The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

    PubMed Central

    Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling

    2015-01-01

    ABSTRACT NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. IMPORTANCE The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice

  3. Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis.

    PubMed

    Gaffaney, Jon D; Dunning, F Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R

    2008-11-14

    Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

  4. Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion*S⃞

    PubMed Central

    Ulijasz, Andrew T.; Cornilescu, Gabriel; von Stetten, David; Kaminski, Steve; Mroginski, Maria Andrea; Zhang, Junrui; Bhaya, Devaki; Hildebrandt, Peter; Vierstra, Richard D.

    2008-01-01

    Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B′, that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. 1H-15N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters. PMID:18480055

  5. Imprinted chromosomal domains revealed by allele-specific replication timing of the GABRB3 and GABRA5 genes

    SciTech Connect

    LaSalle, J.; Flint, A.; Lalande, M.

    1994-09-01

    The GABRB3 and GABRA5 genes are organized as a cluster in chromosome 15q11-q13. The genes are separated by around 100 kb and arranged in opposite transcriptional orientations. The GABA{sub A} receptor cluster lies near the Angelman and Prader-Willi loci and displays asynchronous DNA replication, suggesting that this region is subject to parental imprinting. In order to further study the association between DNA replication and imprinting, allele-specific replication was assayed by fluorescence in situ hybridization with {lambda}-phage probes from the GABRB3/A5 region and a D15Z1 satellite probe to identify the parental origin of each chromosome. The replication kinetics of each allele was determined by using a flow sorter to fractionate mitogen-stimulated lymphocytes on the basis of cell cycle progression prior to FISH analysis. These kinetic studies reveal a 50-150 kb chromosomal domain extending from the middle of the GABRB3/A5 intergenic region into the GABRA5 5{prime}-UTR which displays maternal replication in early S with paternal replication delayed until the end of S. In contrast, genomic regions on either side of this maternal early replication domain exhibit the opposite pattern with paternal before maternal replication and both alleles replicating in the latter half of S. These results indicate that the GABRB3/A5 region is divided into domains in which replication timing is determined by parental origin. In addition to a loss of asynchronous replication, organization into replication timing domains is also lost in lymphocytes from maternal and paternal uniparental disomy 15 patients suggesting that a chromosome contribution from both parents is required for the establishment of the imprinted replication domains.

  6. Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

    PubMed

    Dinesh, Dhurvas Chandrasekaran; Kovermann, Michael; Gopalswamy, Mohanraj; Hellmuth, Antje; Calderón Villalobos, Luz Irina A; Lilie, Hauke; Balbach, Jochen; Abel, Steffen

    2015-05-12

    The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼ 6.4 μM) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

  7. Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking*

    PubMed Central

    Kang, Dong Soo; Kern, Ronald C.; Puthenveedu, Manojkumar A.; von Zastrow, Mark; Williams, John C.; Benovic, Jeffrey L.

    2009-01-01

    Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)2GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis. PMID:19710023

  8. Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

    SciTech Connect

    Kang, D.; Kern, R; Puthenveedu, M; von Zastrow, M; Williams, J; Benovic, J

    2009-01-01

    Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.

  9. Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

    PubMed

    Dym, Orly; Albeck, Shira; Unger, Tamar; Jacobovitch, Jossef; Branzburg, Anna; Michael, Yigal; Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Elbaum, Michael

    2008-08-12

    Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.

  10. Crystal Structure of the APOBEC3G Catalytic Domain Reveals Potential Oligomerization Interfaces

    SciTech Connect

    Shandilya, Shivender M.D.; Nalam, Madhavi N.L.; Nalivaika, Ellen A.; Gross, Phillip J.; Valesano, Johnathan C.; Shindo, Keisuke; Li, Ming; Munson, Mary; Royer, William E.; Harjes, Elena; Kono, Takahide; Matsuo, Hiroshi; Harris, Reuben S.; Somasundaran, Mohan; Schiffer, Celia A.

    2010-02-11

    APOBEC3G is a DNA cytidine deaminase that has antiviral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 {angstrom}. This structure corroborates features previously observed in nuclear magnetic resonance (NMR) studies, a bulge in the second {beta} strand and a lengthening of the second {alpha} helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed, suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.

  11. Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

    SciTech Connect

    Schwarten, Melanie; Stoldt, Matthias; Mohrlueder, Jeannine; Willbold, Dieter

    2010-05-07

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8{sup G116} shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  12. Structure of sorting nexin 11 (SNX11) reveals a novel extended phox homology (PX) domain critical for inhibition of SNX10-induced vacuolation.

    PubMed

    Xu, Jinxin; Xu, Tingting; Wu, Bin; Ye, Yinghua; You, Xiaojuan; Shu, Xiaodong; Pei, Duanqing; Liu, Jinsong

    2013-06-07

    Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

  13. Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) promotes non-homologous end joining and inhibits homologous recombination repair upon DNA damage.

    PubMed

    Tang, Mengfan; Li, Yujing; Zhang, Xiya; Deng, Tingting; Zhou, Zhifen; Ma, Wenbin; Songyang, Zhou

    2014-12-05

    Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Side chain flexibility and coupling between the S4-S5 linker and the TRP domain in thermo-sensitive TRP channels: Insights from protein modeling.

    PubMed

    Romero-Romero, Sergio; Gomez Lagunas, Froylan; Balleza, Daniel

    2017-04-01

    The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.

  15. Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma.

    PubMed

    Decock, Anneleen; Ongenaert, Maté; Cannoodt, Robrecht; Verniers, Kimberly; De Wilde, Bram; Laureys, Geneviève; Van Roy, Nadine; Berbegall, Ana P; Bienertova-Vasku, Julie; Bown, Nick; Clément, Nathalie; Combaret, Valérie; Haber, Michelle; Hoyoux, Claire; Murray, Jayne; Noguera, Rosa; Pierron, Gaelle; Schleiermacher, Gudrun; Schulte, Johannes H; Stallings, Ray L; Tweddle, Deborah A; De Preter, Katleen; Speleman, Frank; Vandesompele, Jo

    2016-01-12

    Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.

  16. Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

    PubMed Central

    Decock, Anneleen; Ongenaert, Maté; Cannoodt, Robrecht; Verniers, Kimberly; De Wilde, Bram; Laureys, Geneviève; Van Roy, Nadine; Berbegall, Ana P.; Bienertova-Vasku, Julie; Bown, Nick; Clément, Nathalie; Combaret, Valérie; Haber, Michelle; Hoyoux, Claire; Murray, Jayne; Noguera, Rosa; Pierron, Gaelle; Schleiermacher, Gudrun; Schulte, Johannes H.; Stallings, Ray L.; Tweddle, Deborah A.; De Preter, Katleen; Speleman, Frank; Vandesompele, Jo

    2016-01-01

    Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature. PMID:26646589

  17. X-Ray Crystal Structure of a TRPM Assembly Domain Reveals An Antiparallel Four-Stranded Coiled-Coil

    SciTech Connect

    Fujiwara, Y.; Minor, D.L.; Jr.

    2009-05-18

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.

  18. Cognitive flexibility in verbal and nonverbal domains and decision making in anorexia nervosa patients: a pilot study

    PubMed Central

    2011-01-01

    Background This paper aimed to investigate cognitive rigidity and decision making impairments in patients diagnosed with Anorexia Nervosa Restrictive type (AN-R), assessing also verbal components. Methods Thirty patients with AN-R were compared with thirty age-matched healthy controls (HC). All participants completed a comprehensive neuropsychological battery comprised of the Trail Making Test, Wisconsin Card Sorting Test, Hayling Sentence Completion Task, and the Iowa Gambling Task. The Beck Depression Inventory was administered to evaluate depressive symptomatology. The influence of both illness duration and neuropsychological variables was considered. Body Mass Index (BMI), years of education, and depression severity were considered as covariates in statistical analyses. Results The AN-R group showed poorer performance on all neuropsychological tests. There was a positive correlation between illness duration and the Hayling Sentence Completion Task Net score, and number of completion answers in part B. There was a partial effect of years of education and BMI on neuropsychological test performance. Response inhibition processes and verbal fluency impairment were not associated with BMI and years of education, but were associated with depression severity. Conclusions These data provide evidence that patients with AN-R have cognitive rigidity in both verbal and non-verbal domains. The role of the impairment on verbal domains should be considered in treatment. Further research is warranted to better understand the relationship between illness state and cognitive rigidity and impaired decision-making. PMID:21982555

  19. Phylogeography of Y-Chromosome Haplogroup I Reveals Distinct Domains of Prehistoric Gene Flow in Europe

    PubMed Central

    Rootsi, Siiri; Magri, Chiara; Kivisild, Toomas; Benuzzi, Giorgia; Help, Hela; Bermisheva, Marina; Kutuev, Ildus; Barać, Lovorka; Peričić, Marijana; Balanovsky, Oleg; Pshenichnov, Andrey; Dion, Daniel; Grobei, Monica; Zhivotovsky, Lev A.; Battaglia, Vincenza; Achilli, Alessandro; Al-Zahery, Nadia; Parik, Jüri; King, Roy; Cinnioğlu, Cengiz; Khusnutdinova, Elsa; Rudan, Pavao; Balanovska, Elena; Scheffrahn, Wolfgang; Simonescu, Maya; Brehm, Antonio; Goncalves, Rita; Rosa, Alexandra; Moisan, Jean-Paul; Chaventre, Andre; Ferak, Vladimir; Füredi, Sandor; Oefner, Peter J.; Shen, Peidong; Beckman, Lars; Mikerezi, Ilia; Terzić, Rifet; Primorac, Dragan; Cambon-Thomsen, Anne; Krumina, Astrida; Torroni, Antonio; Underhill, Peter A.; Santachiara-Benerecetti, A. Silvana; Villems, Richard; Semino, Ornella

    2004-01-01

    To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ∼9,000 years ago. PMID:15162323

  20. Study on hydraulic exciting vibration due to flexible valve in pump system with method of characteristics in the time domain

    NASA Astrophysics Data System (ADS)

    Yu, Y. H.; Liu, D.; Yang, X. F.; Si, J.

    2017-08-01

    To analyse the flow characteristics of leakage as well as the mechanism of selfexcited vibration in valves, the method of characteristics was used to assess the effect of flexible valve leakage on the self-excited vibration in water-supply pump system. Piezometric head in upstream of the valve as a function of time was obtained. Two comparative schemes were proposed to simulate the process of self-excited vibration by changing the length, the material of the pipeline and the leakage of valves in the above pump system. It is shown that the length and material of the pipe significantly affect the amplitude and cycle of self-excited vibration as well as the increasing rate of the vibration amplitude. In addition, the leakage of the valve has little influence on the amplitude and cycle of self-excited vibration, but has a significant effect on the increasing rate of vibration amplitude. A pipe explosion accident may occur without the inhibiting of self-excited vibration.

  1. Identification of Hammerhead Ribozymes in All Domains of Life Reveals Novel Structural Variations

    PubMed Central

    Perreault, Jonathan; Weinberg, Zasha; Roth, Adam; Popescu, Olivia; Chartrand, Pascal; Ferbeyre, Gerardo; Breaker, Ronald R.

    2011-01-01

    Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of “type II” hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred. PMID:21573207

  2. Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.

    PubMed

    Lamboy, Jorge A; Kim, Hajin; Dembinski, Holly; Ha, Taekjip; Komives, Elizabeth A

    2013-07-24

    Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1-4), at ARs 2 and 5 (AR 2-5), and at ARs 3 and 6 (AR 3-6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 "loses grip". AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

  3. Single-Molecule FRET Reveals the Native-State Dynamics of the IκBα Ankyrin Repeat Domain

    PubMed Central

    Lamboy, Jorge A.; Kim, Hajin; Dembinski, Holly; Ha, Taekjip; Komives, Elizabeth A.

    2013-01-01

    Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain. PMID:23619335

  4. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking.

    PubMed

    Pardo, Raúl; Molina-Calavita, Maria; Poizat, Ghislaine; Keryer, Guy; Humbert, Sandrine; Saudou, Frédéric

    2010-06-01

    Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease. We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help

  5. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking

    PubMed Central

    2010-01-01

    Background Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease. Results We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. Conclusion We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its

  6. Structure of the RAG1 nonamer-binding domain with DNA reveals a dimer that mediates DNA synapsis

    PubMed Central

    Fang Yin, Fang; Bailey, Scott; Innis, C. Axel; Ciubotaru, Mihai; Kamtekar, Satwik; Steitz, Thomas A.; Schatz, David G.

    2009-01-01

    The products of recombination activating genes (RAG) 1 and 2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA-binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer-binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1/2. PMID:19396172

  7. Structure of the RAG1 Nonamer Binding Domain with DNA Reveals a Dimer that Mediates DNA Synapsis

    SciTech Connect

    Yin, F.; Bailey, S; Innis, C; Ciubotaru, M; Kamtekar, S; Steitz, T; Schatz, D

    2009-01-01

    The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.

  8. A highly accurate spectral method for the Navier-Stokes equations in a semi-infinite domain with flexible boundary conditions

    NASA Astrophysics Data System (ADS)

    Matsushima, Toshiki; Ishioka, Keiichi

    2017-04-01

    This paper presents a spectral method for numerically solving the Navier-Stokes equations in a semi-infinite domain bounded by a flat plane: the aim is to obtain high accuracy with flexible boundary conditions. The proposed use is for numerical simulations of small-scale atmospheric phenomena near the ground. We introduce basis functions that fit the semi-infinite domain, and an integral condition for vorticity is used to reduce the computational cost when solving the partial differential equations that appear when the viscosity term is treated implicitly. Furthermore, in order to ensure high accuracy, two iteration techniques are applied when solving the system of linear equations and in determining boundary values. This significantly reduces numerical errors, and the proposed method enables high-resolution numerical experiments. This is demonstrated by numerical experiments showing the collision of a vortex ring into a wall; these were performed using numerical models based on the proposed method. It is shown that the time evolution of the flow field is successfully obtained not only near the boundary, but also in a region far from the boundary. The applicability of the proposed method and the integral condition is discussed.

  9. Arrangement of ribosomal genes in nucleolar domains revealed by detection of "Christmas tree" components.

    PubMed

    Mosgoeller, W; Schöfer, C; Steiner, M; Sylvester, J E; Hozák, P

    2001-12-01

    We investigated how the transcribing ribosomal genes ("Christmas trees") of HeLa cells are arranged in the nucleolus. Hypotonic conditions let the granular component disperse, while fibrillar centres and parts of the dense fibrillar component were resistant to low ionic strength conditions. Both remained within the former nucleolar territory. We used immunocytochemistry and in situ hybridisation at the light microscopic and ultrastructural level for the analysis of the internal nucleolar structures. The 5' ends of ribosomal RNA and ribosomal DNA sequences were found associated with the periphery of fibrillar centres. The hypotony-resistant parts of the dense fibrillar component did not contain the 5' end of the transcript or the gene. The downstream ribosomal DNA sequences were found in the nucleolar territory but not associated with any hypotony-resistant structures. The downstream ribosomal RNA revealed a similar distribution. We show that transcription initiation and transcript elongation occur in different molecular and structural environments. Transcription initiation is located at the periphery of fibrillar centres. Evidently the dense fibrillar component is non-homogeneous in molecular composition. Transcript elongation is continued in a part of the dense fibrillar component which is dissolved under intermediate hypotonic conditions. A structural model of nucleolar transcription is suggested.

  10. Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

    PubMed

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2009-04-01

    The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

  11. Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

    PubMed Central

    Gill, Martin B.; Vivithanaporn, Pornpun; Swanson, Geoffrey T.

    2009-01-01

    Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an “open” conformation, or “lock” the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis. PMID:19342380

  12. Flexibility of the Head-Stalk Linker Domain of Paramyxovirus HN Glycoprotein Is Essential for Triggering Virus Fusion

    PubMed Central

    Adu-Gyamfi, Emmanuel; Kim, Lori S.; Jardetzky, Theodore S.

    2016-01-01

    ABSTRACT The Paramyxoviridae comprise a large family of enveloped, negative-sense, single-stranded RNA viruses with significant economic and public health implications. For nearly all paramyxoviruses, infection is initiated by fusion of the viral and host cell plasma membranes in a pH-independent fashion. Fusion is orchestrated by the receptor binding protein hemagglutinin-neuraminidase (HN; also called H or G depending on the virus type) protein and a fusion (F) protein, the latter undergoing a major refolding process to merge the two membranes. Mechanistic details regarding the coupling of receptor binding to F activation are not fully understood. Here, we have identified the flexible loop region connecting the bulky enzymatically active head and the four-helix bundle stalk to be essential for fusion promotion. Proline substitution in this region of HN of parainfluenza virus 5 (PIV5) and Newcastle disease virus HN abolishes cell-cell fusion, whereas HN retains receptor binding and neuraminidase activity. By using reverse genetics, we engineered recombinant PIV5-EGFP viruses with mutations in the head-stalk linker region of HN. Mutations in this region abolished virus recovery and infectivity. In sum, our data suggest that the loop region acts as a “hinge” around which the bulky head of HN swings to-and-fro to facilitate timely HN-mediate F-triggering, a notion consistent with the stalk-mediated activation model of paramyxovirus fusion. IMPORTANCE Paramyxovirus fusion with the host cell plasma membrane is essential for virus infection. Membrane fusion is orchestrated via interaction of the receptor binding protein (HN, H, or G) with the viral fusion glycoprotein (F). Two distinct models have been suggested to describe the mechanism of fusion: these include “the clamp” and the “provocateur” model of activation. By using biochemical and reverse genetics tools, we have obtained strong evidence in favor of the HN stalk-mediated activation of paramyxovirus

  13. Atomic structure of recombinant thaumatin II reveals flexible conformations in two residues critical for sweetness and three consecutive glycine residues.

    PubMed

    Masuda, Tetsuya; Mikami, Bunzo; Tani, Fumito

    2014-11-01

    Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin.

  14. Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking

    PubMed Central

    Militi, Stefania; Maywood, Elizabeth S.; Sandate, Colby R.; Chesham, Johanna E.; Parsons, Michael J.; Vibert, Jennifer L.; Joynson, Greg M.; Partch, Carrie L.; Hastings, Michael H.; Nolan, Patrick M.

    2016-01-01

    The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1–CLOCK complexes is suppressed by PER–CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2Edo/Edo; Csnk1eTau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping. PMID:26903623

  15. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.

    PubMed

    Nelson, Wendy D; Blakely, Sarah E; Nesmelov, Yuri E; Thomas, David D

    2005-03-15

    We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.

  16. High-Throughput Ligand Discovery Reveals a Sitewise Gradient of Diversity in Broadly Evolved Hydrophilic Fibronectin Domains

    PubMed Central

    Woldring, Daniel R.; Holec, Patrick V.; Zhou, Hong; Hackel, Benjamin J.

    2015-01-01

    Discovering new binding function via a combinatorial library in small protein scaffolds requires balance between appropriate mutations to introduce favorable intermolecular interactions while maintaining intramolecular integrity. Sitewise constraints exist in a non-spatial gradient from diverse to conserved in evolved antibody repertoires; yet non-antibody scaffolds generally do not implement this strategy in combinatorial libraries. Despite the fact that biased amino acid distributions, typically elevated in tyrosine, serine, and glycine, have gained wider use in synthetic scaffolds, these distributions are still predominantly applied uniformly to diversified sites. While select sites in fibronectin domains and DARPins have shown benefit from sitewise designs, they have not been deeply evaluated. Inspired by this disparity between diversity distributions in natural libraries and synthetic scaffold libraries, we hypothesized that binders resulting from discovery and evolution would exhibit a non-spatial, sitewise gradient of amino acid diversity. To identify sitewise diversities consistent with efficient evolution in the context of a hydrophilic fibronectin domain, >105 binders to six targets were evolved and sequenced. Evolutionarily favorable amino acid distributions at 25 sites reveal Shannon entropies (range: 0.3–3.9; median: 2.1; standard deviation: 1.1) supporting the diversity gradient hypothesis. Sitewise constraints in evolved sequences are consistent with complementarity, stability, and consensus biases. Implementation of sitewise constrained diversity enables direct selection of nanomolar affinity binders validating an efficient strategy to balance inter- and intra-molecular interaction demands at each site. PMID:26383268

  17. Single-Molecule Studies of the Lysine Riboswitch Reveal Effector Dependent Conformational Dynamics of the Aptamer Domain

    PubMed Central

    Fiegland, Larry R.; Garst, Andrew D.; Batey, Robert T.; Nesbitt, David J.

    2013-01-01

    The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant, as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (KD) for lysine binding of 1.7(5) mM, and opening/closing rate constants of 1.4(3) s−1 and 0.203(7) s−1, respectively. These rates predict an apparent dissociation constant for lysine binding (KD, apparent) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports. PMID:23067368

  18. Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

    PubMed

    Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

    2015-07-10

    Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

  19. Anteroposterior patterning in Xenopus embryos: egg fragment assay system reveals a synergy of dorsalizing and posteriorizing embryonic domains.

    PubMed

    Fujii, Hidefumi; Nagai, Takeharu; Shirasawa, Hiroki; Doi, Jun-ya; Yasui, Kinya; Nishimatsu, Shin-ichirou; Takeda, Hiroyuki; Sakai, Masao

    2002-12-01

    Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ).

  20. A multi-scale model for hair follicles reveals heterogeneous domains driving rapid spatiotemporal hair growth patterning

    PubMed Central

    Wang, Qixuan; Oh, Ji Won; Lee, Hye-Lim; Dhar, Anukriti; Peng, Tao; Ramos, Raul; Guerrero-Juarez, Christian Fernando; Wang, Xiaojie; Zhao, Ran; Cao, Xiaoling; Le, Jonathan; Fuentes, Melisa A; Jocoy, Shelby C; Rossi, Antoni R; Vu, Brian; Pham, Kim; Wang, Xiaoyang; Mali, Nanda Maya; Park, Jung Min; Choi, June-Hyug; Lee, Hyunsu; Legrand, Julien M D; Kandyba, Eve; Kim, Jung Chul; Kim, Moonkyu; Foley, John; Yu, Zhengquan; Kobielak, Krzysztof; Andersen, Bogi; Khosrotehrani, Kiarash; Nie, Qing; Plikus, Maksim V

    2017-01-01

    The control principles behind robust cyclic regeneration of hair follicles (HFs) remain unclear. Using multi-scale modeling, we show that coupling inhibitors and activators with physical growth of HFs is sufficient to drive periodicity and excitability of hair regeneration. Model simulations and experimental data reveal that mouse skin behaves as a heterogeneous regenerative field, composed of anatomical domains where HFs have distinct cycling dynamics. Interactions between fast-cycling chin and ventral HFs and slow-cycling dorsal HFs produce bilaterally symmetric patterns. Ear skin behaves as a hyper-refractory domain with HFs in extended rest phase. Such hyper-refractivity relates to high levels of BMP ligands and WNT antagonists, in part expressed by ear-specific cartilage and muscle. Hair growth stops at the boundaries with hyper-refractory ears and anatomically discontinuous eyelids, generating wave-breaking effects. We posit that similar mechanisms for coupled regeneration with dominant activator, hyper-refractory, and wave-breaker regions can operate in other actively renewing organs. DOI: http://dx.doi.org/10.7554/eLife.22772.001 PMID:28695824

  1. Solution structure of the region 51–160 of human KIN17 reveals an atypical winged helix domain

    PubMed Central

    Carlier, Ludovic; Couprie, Joël; le Maire, Albane; Guilhaudis, Laure; Milazzo-Segalas, Isabelle; Courçon, Marie; Moutiez, Mireille; Gondry, Muriel; Davoust, Daniel; Gilquin, Bernard; Zinn-Justin, Sophie

    2007-01-01

    Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28–50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268–393) only found in higher eukaryotes. Here we report the solution structure of the region 51–160 of human KIN17. We show that this fragment folds into a three-α-helix bundle packed against a three-stranded β-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 310-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51–160 might rather be involved in protein–protein interaction through its conserved surface centered on the 310-helix. PMID:18029424

  2. Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain.

    PubMed

    Xia, Yitian; Shang, Yuan; Zhang, Rongguang; Zhu, Jinwei

    2017-08-10

    The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  3. Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

    PubMed Central

    Cayrou, Christelle; Coulombe, Philippe; Vigneron, Alice; Stanojcic, Slavica; Ganier, Olivier; Peiffer, Isabelle; Rivals, Eric; Puy, Aurore; Laurent-Chabalier, Sabine; Desprat, Romain; Méchali, Marcel

    2011-01-01

    In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5′ and 3′ of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates. PMID:21750104

  4. Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation.

    PubMed

    Shimaoka, Motomu; Xiao, Tsan; Liu, Jin-Huan; Yang, Yuting; Dong, Yicheng; Jun, Chang-Duk; McCormack, Alison; Zhang, Rongguang; Joachimiak, Andrzej; Takagi, Junichi; Wang, Jia-Huai; Springer, Timothy A

    2003-01-10

    The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.

  5. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

    PubMed

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  6. K-core decomposition of a protein domain co-occurrence network reveals lower cancer mutation rates for interior cores.

    PubMed

    Emerson, Arnold I; Andrews, Simeon; Ahmed, Ikhlak; Azis, Thasni Ka; Malek, Joel A

    2015-01-01

    Network biology currently focuses primarily on metabolic pathways, gene regulatory, and protein-protein interaction networks. While these approaches have yielded critical information, alternative methods to network analysis will offer new perspectives on biological information. A little explored area is the interactions between domains that can be captured using domain co-occurrence networks (DCN). A DCN can be used to study the function and interaction of proteins by representing protein domains and their co-existence in genes and by mapping cancer mutations to the individual protein domains to identify signals. The domain co-occurrence network was constructed for the human proteome based on PFAM domains in proteins. Highly connected domains in the central cores were identified using the k-core decomposition technique. Here we show that these domains were found to be more evolutionarily conserved than the peripheral domains. The somatic mutations for ovarian, breast and prostate cancer diseases were obtained from the TCGA database. We mapped the somatic mutations to the individual protein domains and the local false discovery rate was used to identify significantly mutated domains in each cancer type. Significantly mutated domains were found to be enriched in cancer disease pathways. However, we found that the inner cores of the DCN did not contain any of the significantly mutated domains. We observed that the inner core protein domains are highly conserved and these domains co-exist in large numbers with other protein domains. Mutations and domain co-occurrence networks provide a framework for understanding hierarchal designs in protein function from a network perspective. This study provides evidence that a majority of protein domains in the inner core of the DCN have a lower mutation frequency and that protein domains present in the peripheral regions of the k-core contribute more heavily to the disease. These findings may contribute further to drug development.

  7. Characterization of Ribeye Subunits in Zebrafish Hair Cells Reveals That Exogenous Ribeye B-Domain and CtBP1 Localize to the Basal Ends of Synaptic Ribbons

    PubMed Central

    Sheets, Lavinia; Hagen, Matthew W.; Nicolson, Teresa

    2014-01-01

    Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE–the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body. PMID:25208216

  8. Peptide–polymer ligands for a tandem WW-domain, an adaptive multivalent protein–protein interaction: lessons on the thermodynamic fitness of flexible ligands

    PubMed Central

    Koschek, Katharina; Durmaz, Vedat; Krylova, Oxana; Wieczorek, Marek; Gupta, Shilpi; Richter, Martin; Bujotzek, Alexander; Fischer, Christina; Haag, Rainer; Freund, Christian; Weber, Marcus

    2015-01-01

    Summary Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3–9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide–polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide–polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3–2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide–polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding. PMID:26124884

  9. Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands.

    PubMed

    Koschek, Katharina; Durmaz, Vedat; Krylova, Oxana; Wieczorek, Marek; Gupta, Shilpi; Richter, Martin; Bujotzek, Alexander; Fischer, Christina; Haag, Rainer; Freund, Christian; Weber, Marcus; Rademann, Jörg

    2015-01-01

    Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3-9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide-polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide-polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3-2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide-polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

  10. Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases.

    PubMed

    Eiler, Daniel; Lin, Jinzhong; Simonetti, Angelita; Klaholz, Bruno P; Steitz, Thomas A

    2013-09-24

    The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the "articulated lever" mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.

  11. Time-domain parameter identification of aeroelastic loads by forced-vibration method for response of flexible structures subject to transient wind

    NASA Astrophysics Data System (ADS)

    Cao, Bochao

    Slender structures representing civil, mechanical and aerospace systems such as long-span bridges, high-rise buildings, stay cables, power-line cables, high light mast poles, crane-booms and aircraft wings could experience vortex-induced and buffeting excitations below their design wind speeds and divergent self-excited oscillations (flutter) beyond a critical wind speed because these are flexible. Traditional linear aerodynamic theories that are routinely applied for their response prediction are not valid in the galloping, or near-flutter regime, where large-amplitude vibrations could occur and during non-stationary and transient wind excitations that occur, for example, during hurricanes, thunderstorms and gust fronts. The linear aerodynamic load formulation for lift, drag and moment are expressed in terms of aerodynamic functions in frequency domain that are valid for straight-line winds which are stationary or weakly-stationary. Application of the frequency domain formulation is restricted from use in the nonlinear and transient domain because these are valid for linear models and stationary wind. The time-domain aerodynamic force formulations are suitable for finite element modeling, feedback-dependent structural control mechanism, fatigue-life prediction, and above all modeling of transient structural behavior during non-stationary wind phenomena. This has motivated the developing of time-domain models of aerodynamic loads that are in parallel to the existing frequency-dependent models. Parameters defining these time-domain models can be now extracted from wind tunnel tests, for example, the Rational Function Coefficients defining the self-excited wind loads can be extracted using section model tests using the free vibration technique. However, the free vibration method has some limitations because it is difficult to apply at high wind speeds, in turbulent wind environment, or on unstable cross sections with negative aerodynamic damping. In the current

  12. The Instructional Dependency of SNARC Effects Reveals Flexibility of the Space-Magnitude Association of Nonsymbolic and Symbolic Magnitudes.

    PubMed

    Lee, Dasom; Chun, Joohyung; Cho, Soohyun

    2016-05-01

    The Spatial-Numerical Association of Response Codes (SNARC) effect refers to the phenomenon that small versus large numbers are responded to faster in the left versus right side of space, respectively. Using a pairwise comparison task, Shaki et al. found that task instruction influences the pattern of SNARC effects of certain types of magnitudes which are less rigid in their space-magnitude association .The present study examined the generalizability of this instruction effect using pairwise comparison of nonsymbolic and symbolic stimuli within a wide range of magnitudes. We contrasted performance between trials in which subjects were instructed to select the stimulus representing the smaller versus larger magnitude within each pair. We found an instruction-dependent pattern of SNARC effects for both nonsymbolic and symbolic magnitudes. Specifically, we observed a SNARC effect for the "Select Smaller" instruction, but a reverse SNARC effect for the "Select Larger" instruction. Considered together with previous studies, our findings suggest that nonsymbolic magnitudes and relatively large symbolic magnitudes have greater flexibility in their space-magnitude association.

  13. The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

    PubMed

    Hong, Zebin; Nowakowski, Michal; Spronk, Chris; Petersen, Steen V; Andreasen, Peter A; Koźmiński, Wiktor; Mulder, Frans A A; Jensen, Jan K

    2015-03-01

    A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.

  14. Domain Analysis of ArcS, the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System, Reveals Functional Differentiation of Its Two Receiver Domains

    PubMed Central

    Bubendorfer, Sebastian

    2013-01-01

    In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli. PMID:23161031

  15. Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

    PubMed

    Perez, H D; Vilander, L; Andrews, W H; Holmes, R

    1994-09-09

    Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.

  16. Identification of cytoplasmic subdomains that control pH-sensing of the Na+/H+ exchanger (NHE1): pH-maintenance, ATP-sensitive, and flexible loop domains.

    PubMed

    Ikeda, T; Schmitt, B; Pouysségur, J; Wakabayashi, S; Shigekawa, M

    1997-02-01

    To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the

  17. Task Effects Reveal Cognitive Flexibility Responding to Frequency and Predictability: Evidence from Eye Movements in Reading and Proofreading

    PubMed Central

    Schotter, Elizabeth R.; Bicknell, Klinton; Howard, Ian; Levy, Roger; Rayner, Keith

    2014-01-01

    It is well-known that word frequency and predictability affect processing time. These effects change magnitude across tasks, but studies testing this use tasks with different response types (e.g., lexical decision, naming, and fixation time during reading; Schilling, Rayner & Chumbley, 1998), preventing direct comparison. Recently, Kaakinen and Hyönä (2010) overcame this problem, comparing fixation times in reading for comprehension and proofreading, showing that the frequency effect was larger in proofreading than in reading. This result could be explained by readers exhibiting substantial cognitive flexibility, and qualitatively changing how they process words in the proofreading task in a way that magnifies effects of word frequency. Alternatively, readers may not change word processing so dramatically, and instead may perform more careful identification generally, increasing the magnitude of many word processing effects (e.g., both frequency and predictability). We tested these possibilities with two experiments: subjects read for comprehension and then proofread for spelling errors (letter transpositions) that produce nonwords (e.g., trcak for track as in Kaakinen & Hyönä) or that produce real but unintended words (e.g., trial for trail) to compare how the task changes these effects. Replicating Kaakinen and Hyönä, frequency effects increased during proofreading. However, predictability effects only increased when integration with the sentence context was necessary to detect errors (i.e., when spelling errors produced words that were inappropriate in the sentence; trial for trail). The results suggest that readers adopt sophisticated word processing strategies to accommodate task demands. PMID:24434024

  18. Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component

    PubMed Central

    Märtlbauer, E.

    2016-01-01

    The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex. PMID:27768742

  19. Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope.

    PubMed

    Mayer, Christina; Slater, Leanne; Erat, Michele C; Konrat, Robert; Vakonakis, Ioannis

    2012-03-02

    Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

  20. Structure of the Lectin Regulatory Domain of the Cholesterol-Dependent Cytolysin Lectinolysin Reveals the Basis for Its Lewis Antigen Specificity

    PubMed Central

    Feil, Susanne C.; Lawrence, Sara; Mulhern, Terrence D.; Holien, Jessica K.; Hotze, Eileen M.; Farrand, Stephen; Tweten, Rodney K.; Parker, Michael W.

    2013-01-01

    SUMMARY The cholesterol-dependent cytolysins (CDCs) punch holes in target cell membranes through a highly regulated process. Streptococcus mitis lectinolysin (LLY) exhibits another layer of regulation with a lectin domain that enhances the pore-forming activity of the toxin. We have determined the crystal structures of the lectin domain by itself and in complex with various glycans that reveal the molecular basis for the Lewis antigen specificity of LLY. A small-angle X-ray scattering study of intact LLY reveals the molecule is flat and elongated with the lectin domain oriented so that the Lewis antigen-binding site is exposed. We suggest that the lectin domain enhances the pore-forming activity of LLY by concentrating toxin molecules at fucose-rich sites on membranes, thus promoting the formation of pre-pore oligomers on the surface of susceptible cells. PMID:22325774

  1. Just a Flexible Linker? The Structural and Dynamic Properties of CBP-ID4 Revealed by NMR Spectroscopy

    PubMed Central

    Piai, Alessandro; Calçada, Eduardo O.; Tarenzi, Thomas; Grande, Alessandro del; Varadi, Mihaly; Tompa, Peter; Felli, Isabella C.; Pierattelli, Roberta

    2016-01-01

    Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2–25 and 101–128 (1852–1875 and 1951–1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners. PMID:26789760

  2. Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3′–5′ exonuclease activity

    PubMed Central

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2009-01-01

    The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3′–5′ exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-dependent 3′–5′ exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn2+-dependent 3′–5′ exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3′–5′ exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3′–5′ exonuclease activity. PMID:19211662

  3. Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family

    PubMed Central

    Trempe, Jean-François; Šašková, Klára Grantz; Sivá, Monika; Ratcliffe, Colin D. H.; Veverka, Václav; Hoegl, Annabelle; Ménade, Marie; Feng, Xin; Shenker, Solomon; Svoboda, Michal; Kožíšek, Milan; Konvalinka, Jan; Gehring, Kalle

    2016-01-01

    The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains. PMID:27646017

  4. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    SciTech Connect

    Zhang, Bing; Liu, Caini; Qian, Wen; Han, Yue; Li, Xiaoxia; Deng, Junpeng

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  5. The solution structure of the periplasmic domain of the TonB system ExbD protein reveals an unexpected structural homology with siderophore-binding proteins.

    PubMed

    Garcia-Herrero, Alicia; Peacock, R Sean; Howard, S Peter; Vogel, Hans J

    2007-11-01

    The transport of iron complexes through outer membrane transporters from Gram-negative bacteria is highly dependent on the TonB system. Together, the three components of the system, TonB, ExbB and ExbD, energize the transport of iron complexes through the outer membrane by utilizing the proton motive force across the cytoplasmic membrane. The three-dimensional (3D) structure of the periplasmic domain of TonB has previously been determined. However, no detailed structural information for the other two components of the TonB system is currently available and their role in the iron-uptake process is not yet clearly understood. ExbD from Escherichia coli contains 141 residues distributed in three domains: a small N-terminal cytoplasmic region, a single transmembrane helix and a C-terminal periplasmic domain. Here we describe the first well-defined solution structure of the periplasmic domain of ExbD (residues 44-141) solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric structure presents three clearly distinct regions: an N-terminal flexible tail (residues 44-63), a well-defined folded region (residues 64-133) followed by a small C-terminal flexible region (residues 134-141). The folded region is formed by two alpha-helices that are located on one side of a single beta-sheet. The central beta-sheet is composed of five beta-strands, with a mixed parallel and antiparallel arrangement. Unexpectedly, this fold closely resembles that found in the C-terminal lobe of the siderophore-binding proteins FhuD and CeuE. The ExbD periplasmic domain has a strong tendency to aggregate in vitro and 3D-TROSY (transverse relaxation optimized spectroscopy) NMR experiments of the deuterated protein indicate that the multimeric protein has nearly identical secondary structure to that of the monomeric form. Chemical shift perturbation studies suggest that the Glu-Pro region (residues 70-83) of TonB can bind weakly to the surface and the flexible C

  6. History of Mexican Easel Paintings from an Altarpiece Revealed by Non-invasive Terahertz Time-Domain Imaging

    NASA Astrophysics Data System (ADS)

    Gomez-Sepulveda, A. M.; Hernandez-Serrano, A. I.; Radpour, R.; Koch-Dandolo, C. L.; Rojas-Landeros, S. C.; Ascencio-Rojas, L. F.; Zarate, Alvaro; Hernandez, Gerardo; Gonzalez-Tirado, R. C.; Insaurralde-Caballero, M.; Castro-Camus, E.

    2017-04-01

    Four easel paintings attributed to Hermenegildo Bustos ( Purísima del Rincón, Guanajuato, Mexico), one of the most renowned painters of the late nineteenth and early twentieth century Mexican art, have been investigated by means of terahertz time-domain imaging (THz-TDI) and standard imaging techniques, such as near-IR reflectography and X-ray radiography. The archival sources and the recent studies on the paintings suggest that the artworks were created in the eighteenth century and underwent several modifications since then until the intervention of Bustos who authored the currently visible depictions. By combining the records of the paintings obtained by imaging with the different methodologies, aspects of the previous depictions and further details on the paintings' history have been revealed, with THz-TDI playing a key role in attributing a chronological evolution of the images. The paintings of Purísima are the first THz-TDI-scanned paintings belonging to the Mexican cultural heritage.

  7. Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly.

    PubMed

    Gamalinda, Michael; Woolford, John L

    2014-11-01

    Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function. © 2014 Gamalinda and Woolford; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Structural Studies of the Alzheimer's Amyloid Precursor Protein Copper-Binding Domain Reveal How It Binds Copper Ions

    SciTech Connect

    Kong, G.K.-W.; Adams, J.J.; Harris, H.H.; Boas, J.F.; Curtain, C.C.; Galatis, D.; Master, C.L.; Barnham, K.J.; McKinstry, W.J.; Cappai, R.; Parker, M.W.; /Sydney U. /Monash U. /Melbourne U.

    2007-07-09

    Alzheimer's disease (AD) is the major cause of dementia. Amyloid {beta} peptide (A {beta}), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces A{beta} levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu[2+]-bound CuBD reveals that the metal ligands are His147, His151, Tyrl68 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu[+]-bound CuBD is almost identical to the Cu[2+]-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu[+], thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins.

  9. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

    SciTech Connect

    Baram, David; Pyetan, Erez; Sittner, Assa; Auerbach-Nevo, Tamar; Bashan, Anat; Yonath, Ada

    2010-07-13

    Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-{angstrom} crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its 'signature motif' as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

  10. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action.

    PubMed

    Baram, David; Pyetan, Erez; Sittner, Assa; Auerbach-Nevo, Tamar; Bashan, Anat; Yonath, Ada

    2005-08-23

    Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-A crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its "signature motif" as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

  11. Hyperreflective dots surrounding the central retinal artery and vein in optic disc melanocytoma revealed by spectral domain optical coherence tomography.

    PubMed

    Okubo, Akiko; Unoki, Kazuhiko; Yoshikawa, Hiroshi; Ishibashi, Tatsuro; Sameshima, Munefumi; Sakamoto, Taiji

    2013-01-01

    To report findings of optic disc melanocytoma (ODM) obtained using spectral domain optical coherence tomography (SD OCT), with special reference to the central retinal artery and vein surrounded by hyperreflective dots. Retrospective review of five eyes of five patients with ODM. Demographic information, ophthalmic examination including best-corrected visual acuity, dilated funduscopic examination, and SD OCT images were evaluated. Dome-shaped, darkly pigmented tumors were seen ophthalmoscopically in the optic discs of all eyes. On OCT, the first branches of the central retinal artery and/or vein were well defined as oblique sections of tubular structures with a perivascular distribution of hyperreflective dots in the elevated retina (nerve fiber layer) over the tumor. The portions where these vessels turn toward the retina were displaced more anteriorly than those of eyes without ODM. Hyperreflective dots of various sizes were also observed in elevated retinas over the tumors, which shadowed and obscured the subjacent tissue in all eyes. SD OCT provides higher definition images of ODM relating to the branches of the central retinal artery/vein, revealing anterior displacement of vessels and perivascular distribution of hyperreflective dots that suggest melanophages and/or tumor cells or proteins and/or lipid deposits.

  12. Retinal Structure of Birds of Prey Revealed by Ultra-High Resolution Spectral-Domain Optical Coherence Tomography

    PubMed Central

    Ruggeri, Marco; Major, James C.; McKeown, Craig; Knighton, Robert W.; Puliafito, Carmen A.

    2010-01-01

    Purpose. To reveal three-dimensional (3-D) information about the retinal structures of birds of prey in vivo. Methods. An ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) system was built for in vivo imaging of retinas of birds of prey. The calibrated imaging depth and axial resolution of the system were 3.1 mm and 2.8 μm (in tissue), respectively. 3-D segmentation was performed for calculation of the retinal nerve fiber layer (RNFL) map. Results. High-resolution OCT images were obtained of the retinas of four species of birds of prey: two diurnal hawks (Buteo platypterus and Buteo brachyurus) and two nocturnal owls (Bubo virginianus and Strix varia). These images showed the detailed retinal anatomy, including the retinal layers and the structure of the deep and shallow foveae. The calculated thickness map showed the RNFL distribution. Traumatic injury to one bird's retina was also successfully imaged. Conclusions. Ultra-high resolution SD-OCT provides unprecedented high-quality 2-D and 3-D in vivo visualization of the retinal structures of birds of prey. SD-OCT is a powerful imaging tool for vision research in birds of prey. PMID:20554605

  13. Retinal structure of birds of prey revealed by ultra-high resolution spectral-domain optical coherence tomography.

    PubMed

    Ruggeri, Marco; Major, James C; McKeown, Craig; Knighton, Robert W; Puliafito, Carmen A; Jiao, Shuliang

    2010-11-01

    To reveal three-dimensional (3-D) information about the retinal structures of birds of prey in vivo. An ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) system was built for in vivo imaging of retinas of birds of prey. The calibrated imaging depth and axial resolution of the system were 3.1 mm and 2.8 μm (in tissue), respectively. 3-D segmentation was performed for calculation of the retinal nerve fiber layer (RNFL) map. High-resolution OCT images were obtained of the retinas of four species of birds of prey: two diurnal hawks (Buteo platypterus and Buteo brachyurus) and two nocturnal owls (Bubo virginianus and Strix varia). These images showed the detailed retinal anatomy, including the retinal layers and the structure of the deep and shallow foveae. The calculated thickness map showed the RNFL distribution. Traumatic injury to one bird's retina was also successfully imaged. Ultra-high resolution SD-OCT provides unprecedented high-quality 2-D and 3-D in vivo visualization of the retinal structures of birds of prey. SD-OCT is a powerful imaging tool for vision research in birds of prey.

  14. History of Mexican Easel Paintings from an Altarpiece Revealed by Non-invasive Terahertz Time-Domain Imaging

    NASA Astrophysics Data System (ADS)

    Gomez-Sepulveda, A. M.; Hernandez-Serrano, A. I.; Radpour, R.; Koch-Dandolo, C. L.; Rojas-Landeros, S. C.; Ascencio-Rojas, L. F.; Zarate, Alvaro; Hernandez, Gerardo; Gonzalez-Tirado, R. C.; Insaurralde-Caballero, M.; Castro-Camus, E.

    2016-12-01

    Four easel paintings attributed to Hermenegildo Bustos (Purísima del Rincón, Guanajuato, Mexico), one of the most renowned painters of the late nineteenth and early twentieth century Mexican art, have been investigated by means of terahertz time-domain imaging (THz-TDI) and standard imaging techniques, such as near-IR reflectography and X-ray radiography. The archival sources and the recent studies on the paintings suggest that the artworks were created in the eighteenth century and underwent several modifications since then until the intervention of Bustos who authored the currently visible depictions. By combining the records of the paintings obtained by imaging with the different methodologies, aspects of the previous depictions and further details on the paintings' history have been revealed, with THz-TDI playing a key role in attributing a chronological evolution of the images. The paintings of Purísima are the first THz-TDI-scanned paintings belonging to the Mexican cultural heritage.

  15. Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.

    PubMed

    Ray, Partho Sarothi; Fox, Paul L

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering "missing links" in the tree of life.

  16. Origin and Evolution of Glutamyl-prolyl tRNA Synthetase WHEP Domains Reveal Evolutionary Relationships within Holozoa

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. PMID:24968216

  17. Genome-Wide Analysis of PDZ Domain Binding Reveals Inherent Functional Overlap within the PDZ Interaction Network

    PubMed Central

    te Velthuis, Aartjan J. W.; Sakalis, Philippe A.; Fowler, Donald A.; Bagowski, Christoph P.

    2011-01-01

    Binding selectivity and cross-reactivity within one of the largest and most abundant interaction domain families, the PDZ family, has long been enigmatic. The complete human PDZ domain complement (the PDZome) consists of 267 domains and we applied here a Bayesian selectivity model to predict hundreds of human PDZ domain interactions, using target sequences of 22,997 non-redundant proteins. Subsequent analysis of these binding scores shows that PDZs can be divided into two genome-wide clusters that coincide well with the division between canonical class 1 and 2 PDZs. Within the class 1 PDZs we observed binding overlap at unprecedented levels, mediated by two residues at positions 1 and 5 of the second α-helix of the binding pocket. Eight PDZ domains were subsequently selected for experimental binding studies and to verify the basics of our predictions. Overall, the PDZ domain class 1 cross-reactivity identified here implies that auxiliary mechanisms must be in place to overcome this inherent functional overlap and to minimize cross-selectivity within the living cell. Indeed, when we superimpose PDZ domain binding affinities with gene ontologies, network topology data and the domain position within a PDZ superfamily protein, functional overlap is minimized and PDZ domains position optimally in the binding space. We therefore propose that PDZ domain selectivity is achieved through cellular context rather than inherent binding specificity. PMID:21283644

  18. Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry.

    PubMed

    Snyder, Greg A; Deredge, Daniel; Waldhuber, Anna; Fresquez, Theresa; Wilkins, David Z; Smith, Patrick T; Durr, Susi; Cirl, Christine; Jiang, Jiansheng; Jennings, William; Luchetti, Timothy; Snyder, Nathaniel; Sundberg, Eric J; Wintrode, Patrick; Miethke, Thomas; Xiao, T Sam

    2014-01-10

    The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys(89) and Cys(134). A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.

  19. Crystal Structures of the Toll/Interleukin-1 Receptor (TIR) Domains from the Brucella Protein TcpB and Host Adaptor TIRAP Reveal Mechanisms of Molecular Mimicry*

    PubMed Central

    Snyder, Greg A.; Deredge, Daniel; Waldhuber, Anna; Fresquez, Theresa; Wilkins, David Z.; Smith, Patrick T.; Durr, Susi; Cirl, Christine; Jiang, Jiansheng; Jennings, William; Luchetti, Timothy; Snyder, Nathaniel; Sundberg, Eric J.; Wintrode, Patrick; Miethke, Thomas; Xiao, T. Sam

    2014-01-01

    The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys89 and Cys134. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP. PMID:24275656

  20. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication

    PubMed Central

    Carr, Stephen B.; Phillips, Simon E.V.; Thomas, Christopher D.

    2016-01-01

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. PMID:26792891

  1. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication.

    PubMed

    Carr, Stephen B; Phillips, Simon E V; Thomas, Christopher D

    2016-03-18

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

    PubMed

    Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

    2017-07-28

    Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis.

    PubMed

    Dai, Yue; Haque, Mohammad Mahfuzul; Stuehr, Dennis J

    2017-04-21

    The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the FMN domain is thought to be essential for the electron transfer (ET) reactions in NOSs. To directly examine this concept, we utilized a "Cys-lite" neuronal NOS flavoprotein domain and substituted Cys for two residues (Glu-816 and Arg-1229) forming a salt bridge between the NADPH/FAD and FMN domains in the conformationally closed structure to allow cross-domain disulfide bond formation or cross-linking by bismaleimides of various lengths. The disulfide bond cross-link caused a ≥95% loss of cytochrome c reductase activity that was reversible with DTT treatment, whereas graded cross-link lengthening gradually increased activity, thus defining the conformational constraints in the catalytic process. We used spectroscopic and stopped-flow techniques to further investigate how the changes in FMN domain conformational freedom impact the following: (i) the NADPH interaction; (ii) kinetics of electron loading (flavin reduction); (iii) stabilization of open versus closed conformational forms in two different flavin redox states; (iv) reactivity of the reduced FMN domain toward cytochrome c; (v) response to calmodulin binding; and (vi) the rates of interflavin ET and the FMN domain conformational dynamics. Together, our findings help explain how the spatial and temporal behaviors of the FMN domain impact catalysis by the NOS flavoprotein domain and how these behaviors are governed to enable electron flow through the enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    SciTech Connect

    Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa; Leybourne, Matthew; Grishin, Nick V.; Zhang, Hong

    2009-01-26

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric 'lids' that

  5. Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype

    DOE PAGES

    Traller, Jesse C.; Cokus, Shawn J.; Lopez, David A.; ...

    2016-11-25

    Here, improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids. As a result, we sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction,more » and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches. Furthermore, functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.« less

  6. Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain

    SciTech Connect

    Miller, D.J.; Ouellette, N.; Evodokimova, E.; Savchenko, A.; Edwards, A.; Anderson, W.F.

    2010-03-08

    S-adenosyl-L-methionine-dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S-adenosyl-L-homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co-crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate-binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S{sup +}-CH{sub 3} bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.

  7. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    SciTech Connect

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  8. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  9. THE STRUCTURES OF COILED-COIL DOMAINS FROM TYPE THREE SECRETION SYSTEM TRANSLOCATORS REVEAL HOMOLOGY TO PORE-FORMING TOXINS

    PubMed Central

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-01-01

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SS) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) which is responsible for over one million deaths per year. The Shigella type III secretion apparatus (T3SA) is comprised of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are comprised of extended length (114 Å in IpaB and 71 Å in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably colicin Ia. This suggests that these mechanistically-separate and functionally-distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events. PMID:22321794

  10. The 1.59Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop.

    PubMed

    Raghunathan, Kannan; Vago, Frank S; Grindem, David; Ball, Terry; Wedemeyer, William J; Bagdasarian, Michael; Arvidson, Dennis N

    2014-02-01

    The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

  11. Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

    PubMed Central

    Moran, Sean D.; Woys, Ann Marie; Buchanan, Lauren E.; Bixby, Eli; Decatur, Sean M.; Zanni, Martin T.

    2012-01-01

    The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. PMID:22328156

  12. Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils.

    PubMed

    Moran, Sean D; Woys, Ann Marie; Buchanan, Lauren E; Bixby, Eli; Decatur, Sean M; Zanni, Martin T

    2012-02-28

    The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.

  13. The Solution Structure of the N-Terminal Domain of Human Tubulin Binding Cofactor C Reveals a Platform for Tubulin Interaction

    PubMed Central

    Garcia-Mayoral, Mª Flor; Castaño, Raquel; Fanarraga, Monica L.; Zabala, Juan Carlos; Rico, Manuel; Bruix, Marta

    2011-01-01

    Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers. PMID:22028797

  14. Crystal structure of the human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity.

    PubMed

    Solomon, Isaac H; Hager, Janet M; Safi, Rachid; McDonnell, Donald P; Redinbo, Matthew R; Ortlund, Eric A

    2005-12-16

    The DNA-binding and ligand-binding functions of nuclear receptors are localized to independent domains separated by a flexible hinge. The DNA-binding domain (DBD) of the human liver receptor homologue-1 (hLRH-1), which controls genes central to development and metabolic homeostasis, interacts with monomeric DNA response elements and contains an Ftz-F1 motif that is unique to the NR5A nuclear receptor subfamily. Here, we present the 2.2A resolution crystal structure of the hLRH-1 DBD in complex with duplex DNA, and elucidate the sequence-specific DNA contacts essential for the ability of LRH-1 to bind to DNA as a monomer. We show that the unique Ftz-F1 domain folds into a novel helix that packs against the DBD but does not contact DNA. Mutations expected to disrupt the positioning of the Ftz-F1 helix do not eliminate DNA binding but reduce the transcriptional activity of full-length LRH-1 significantly. Moreover, we find that altering the Ftz-F1 helix positioning eliminates the enhancement of LRH-1-mediated transcription by the coactivator GRIP1, an action that is associated primarily with the distantly located ligand-binding domain (LBD). Taken together, these results indicate that subtle structural changes in a nuclear receptor DBD can exert long-range functional effects on the LBD of a receptor, and significantly impact transcriptional regulation.

  15. Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding.

    PubMed Central

    Segelke, B. W.; Forstner, M.; Knapp, M.; Trakhanov, S. D.; Parkin, S.; Newhouse, Y. M.; Bellamy, H. D.; Weisgraber, K. H.; Rupp, B.

    2000-01-01

    An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association. PMID:10850798

  16. Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor

    PubMed Central

    Ortega-Roldan, Jose Luis; Ossa, Felipe; Amin, Nader T.; Schnell, Jason R.

    2015-01-01

    The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation. PMID:25647032

  17. Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

    PubMed Central

    Ciaccio, Mark F.; Chuu, Chih-pin; Jones, Richard B.

    2012-01-01

    First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This

  18. The crystal structure of the human nascent polypeptide-associated complex domain reveals a nucleic acid-binding region on the NACA subunit .

    PubMed

    Liu, Yiwei; Hu, Yingxia; Li, Xu; Niu, Liwen; Teng, Maikun

    2010-04-06

    In archaea and eukaryotes, the nascent polypeptide-associated complex (NAC) is one of the cytosolic chaperones that contact the nascent polypeptide chains as they emerge from the ribosome and assist in post-translational processes. The eukaryotic NAC is a heterodimer, and its two subunits form a stable complex through a dimerizing domain called the NAC domain. In addition to acting as a protein translation chaperone, the NAC subunits also function individually in transcriptional regulation. Here we report the crystal structure of the human NAC domain, which reveals the manner of human NAC dimerization. On the basis of the structure, we identified a region in the NAC domain of the human NAC alpha-subunit as a new nucleic acid-binding region, which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the beta-subunit.

  19. The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement in transcriptional regulation

    PubMed Central

    Das, Debanu; Grishin, Nick V.; Kumar, Abhinav; Carlton, Dennis; Bakolitsa, Constantina; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Burra, Prasad; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grzechnik, Anna; Grzechnik, Slawomir K.; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention. PMID:20944208

  20. Strong functional patterns in the evolution of eukaryotic genomes revealed by the reconstruction of ancestral protein domain repertoires

    PubMed Central

    2011-01-01

    Background Genome size and complexity, as measured by the number of genes or protein domains, is remarkably similar in most extant eukaryotes and generally exhibits no correlation with their morphological complexity. Underlying trends in the evolution of the functional content and capabilities of different eukaryotic genomes might be hidden by simultaneous gains and losses of genes. Results We reconstructed the domain repertoires of putative ancestral species at major divergence points, including the last eukaryotic common ancestor (LECA). We show that, surprisingly, during eukaryotic evolution domain losses in general outnumber domain gains. Only at the base of the animal and the vertebrate sub-trees do domain gains outnumber domain losses. The observed gain/loss balance has a distinct functional bias, most strikingly seen during animal evolution, where most of the gains represent domains involved in regulation and most of the losses represent domains with metabolic functions. This trend is so consistent that clustering of genomes according to their functional profiles results in an organization similar to the tree of life. Furthermore, our results indicate that metabolic functions lost during animal evolution are likely being replaced by the metabolic capabilities of symbiotic organisms such as gut microbes. Conclusions While protein domain gains and losses are common throughout eukaryote evolution, losses oftentimes outweigh gains and lead to significant differences in functional profiles. Results presented here provide additional arguments for a complex last eukaryotic common ancestor, but also show a general trend of losses in metabolic capabilities and gain in regulatory complexity during the rise of animals. PMID:21241503

  1. Crystal structure of the trithorax group protein ASH2L reveals a forkhead-like DNA binding domain

    SciTech Connect

    Sarvan, Sabina; Avdic, Vanja; Tremblay, Véronique; Chaturvedi, Chandra-Prakash; Zhang, Pamela; Lanouette, Sylvain; Blais, Alexandre; Brunzelle, Joseph S; Brand, Marjorie; Couture, Jean-François

    2012-05-02

    Absent, small or homeotic discs-like 2 (ASH2L) is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that ASH2L binds DNA using a forkhead-like helix-wing-helix (HWH) domain. In vivo, the ASH2L HWH domain is required for binding to the {beta}-globin locus control region, histone H3 Lys4 (H3K4) trimethylation and maximal expression of the {beta}-globin gene (Hbb-1), validating the functional importance of the ASH2L DNA binding domain.

  2. Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins.

    PubMed

    Martin, Andreas; Schmid, Franz X

    2003-05-09

    The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and

  3. Structures of the Sgt2/SGTA dimerization domain with the Get5/UBL4A UBL domain reveal a novel interaction that forms a conserved dynamic interface

    PubMed Central

    Chartron, Justin W.; VanderVelde, David G.; Clemons, William M.

    2013-01-01

    SUMMARY In the cytoplasm, the correct delivery of membrane proteins is an essential and highly regulated process. The post-translational targeting of the important tail-anchor membrane (TA) proteins has recently been under intense investigation. A specialized pathway, called the GET pathway in yeast and the TRC pathway in vertebrates, recognizes ER targeted TA proteins and delivers them through a complex series of handoffs. An early step is the formation of a complex between Sgt2/SGTA, a co-chaperone with a presumed ubiquitin-like-binding domain (UBD), and Get5/UBL4A, a ubiquitin-like domain (UBL) containing protein. We structurally characterize this novel UBD/UBL interaction for both the yeast and human proteins. This is supported by biophysical studies that demonstrate that complex formation is mediated by electrostatics generating an interface that has high-affinity with rapid kinetics. In total, this work provides a refined model of the interplay of Sgt2 homologs in TA targeting. PMID:23142665

  4. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  5. TIR-domain-containing protein repertoire of nine anthozoan species reveals coral-specific expansions and uncharacterized proteins.

    PubMed

    Poole, Angela Z; Weis, Virginia M

    2014-10-01

    The intracellular toll/interleukin-1 receptor (TIR) domain plays an important role in vertebrate immunity, but the evolution and function of invertebrate TIR-domain-containing proteins is not fully understood. This study characterized and compared the TIR-domain-containing protein repertoire of nine cnidarians in class Anthozoa. A diverse set of proteins, including MyD88 (myeloid differentiation primary response protein 88), toll-like receptor (TLR)-like, interleukin-1 receptor (IL-1R)-like, and TIR-only proteins are present in the species surveyed. Increased numbers of TIR-only proteins were observed in corals compared to anemones, especially in the Acroporid and Pocilloporid coral families. This expansion could be linked to diversity of the microbial community on or in hosts and managing both positive and negative associations. Phylogenetic analysis indicates there are two groups of proteins with IL-1R-like domain architecture in anthozoans that potentially evolved independently of the vertebrate family. Bacterial-like TIR_2 domain proteins are also present, including one sequence with novel domain architecture. Overall this work promotes a better understanding of the anthozoan immune repertoire, which is important in the context learning about ancestral immune pathways and host-microbe interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Bioinformatic analysis of RecQ4 helicases reveals the presence of a RQC domain and a Zn knuckle.

    PubMed

    Marino, Francesca; Vindigni, Alessandro; Onesti, Silvia

    2013-01-01

    RecQ helicases play essential roles in the maintenance of genome stability and contain a highly conserved helicase region generally followed by a characteristic RecQ-C-terminal (RQC) domain, plus a number of variable associated domains. Notable exceptions are the RecQ4 helicases, where none of these additional regions have been described. Particularly striking was the fact that no RQC domain had been reported, considering that the RQC domain had been shown to play an essential role in the catalytic mechanism of most RecQ family members. Here we present the results of detailed bioinformatic analyses of RecQ4 proteins that identify, for the first time, the presence of a putative RQC domain, including some of the key residues involved in DNA binding and unwinding. We also describe the presence of a novel "Zn knuckle" domain, as well as an additional Sld2-homology region, providing new insights into the architecture, function and evolution of these enzymes.

  7. Genomewide analysis of the lateral organ boundaries domain gene family in Eucalyptus grandis reveals members that differentially impact secondary growth.

    PubMed

    Lu, Qiang; Shao, Fenjuan; Macmillan, Colleen; Wilson, Iain W; van der Merwe, Karen; Hussey, Steven G; Myburg, Alexander A; Dong, Xiaomei; Qiu, Deyou

    2017-05-12

    Lateral Organ Boundaries Domain (LBD) proteins are plant-specific transcription factors playing crucial roles in growth and development. However, the function of LBD proteins in Eucalyptus grandis remains largely unexplored. In this study, LBD genes in E. grandis were identified and characterized using bioinformatics approaches. Gene expression patterns in various tissues and the transcriptional responses of EgLBDs to exogenous hormones were determined by qRT-PCR. Functions of the selected EgLBDs were studied by ectopically overexpressing in a hybrid poplar (Populus alba × Populus glandulosa). Expression levels of genes in the transgenic plants were investigated by RNA-seq. Our results showed that there were forty-six EgLBD members in the E. grandis genome and three EgLBDs displayed xylem- (EgLBD29) or phloem-preferential expression (EgLBD22 and EgLBD37). Confocal microscopy indicated that EgLBD22, EgLBD29 and EgLBD37 were localized to the nucleus. Furthermore, we found that EgLBD22, EgLBD29 and EgLBD37 were responsive to the treatments of indol-3-acetic acid and gibberellic acid. More importantly, we demonstrated EgLBDs exerted different influences on secondary growth. Namely, 35S::EgLBD37 led to significantly increased secondary xylem, 35S::EgLBD29 led to greatly increased phloem fibre production, and 35S::EgLBD22 showed no obvious effects. We revealed that key genes related to gibberellin, ethylene and auxin signalling pathway as well as cell expansion were significantly up- or down-regulated in transgenic plants. Our new findings suggest that LBD genes in E. grandis play important roles in secondary growth. This provides new mechanisms to increase wood or fibre production. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  8. The structure of cardiac troponin C regulatory domain with bound Cd2+ reveals a closed conformation and unique ion coordination.

    PubMed

    Zhang, Xiaolu Linda; Tibbits, Glen F; Paetzel, Mark

    2013-05-01

    The amino-terminal domain of cardiac troponin C (cNTnC) is an essential Ca(2+) sensor found in cardiomyocytes. It undergoes a conformational change upon Ca(2+) binding and transduces the signal to the rest of the troponin complex to initiate cardiac muscle contraction. Two classical EF-hand motifs (EF1 and EF2) are present in cNTnC. Under physiological conditions, only EF2 binds Ca(2+); EF1 is a vestigial site that has lost its function in binding Ca(2+) owing to amino-acid sequence changes during evolution. Proteins with EF-hand motifs are capable of binding divalent cations other than calcium. Here, the crystal structure of wild-type (WT) human cNTnC in complex with Cd(2+) is presented. The structure of Cd(2+)-bound cNTnC with the disease-related mutation L29Q, as well as a structure with the residue differences D2N, V28I, L29Q and G30D (NIQD), which have been shown to have functional importance in Ca(2+) sensing at lower temperatures in ectothermic species, have also been determined. The structures resemble the overall conformation of NMR structures of Ca(2+)-bound cNTnC, but differ significantly from a previous crystal structure of Cd(2+)-bound cNTnC in complex with deoxycholic acid. The subtle structural changes observed in the region near the mutations may play a role in the increased Ca(2+) affinity. The 1.4 Å resolution WT cNTnC structure, which is the highest resolution structure yet obtained for cardiac troponin C, reveals a Cd(2+) ion coordinated in the canonical pentagonal bipyramidal geometry in EF2 despite three residues in the loop being disordered. A Cd(2+) ion found in the vestigial ion-binding site of EF1 is coordinated in a noncanonical `distorted' octahedral geometry. A comparison of the ion coordination observed within EF-hand-containing proteins for which structures have been solved in the presence of Cd(2+) is presented. A refolded WT cNTnC structure is also presented.

  9. Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis.

    PubMed

    Wood, Z A; Poole, L B; Karplus, P A

    2001-04-03

    AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.

  10. Two high-resolution structures of potato endo-1,3-β-glucanase reveal subdomain flexibility with implications for substrate binding.

    PubMed

    Wojtkowiak, Agnieszka; Witek, Kamil; Hennig, Jacek; Jaskolski, Mariusz

    2012-06-01

    Endo-1,3-β-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted β-1,3-linked glucosyl residues. The plant enzymes belong to glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. X-ray diffraction data were collected to 1.40 and 1.26 Å resolution from two crystals of endo-1,3-β-glucanase from Solanum tuberosum (potato, cultivar Désirée) which, despite having a similar packing framework, represented two separate crystal forms. In particular, they differed in the Matthews coefficient and are consequently referred to as higher density (HD; 1.40 Å resolution) and lower density (LD; 1.26 Å resolution) forms. The general fold of the protein resembles that of other known plant endo-1,3-β-glucanases and is defined by a (β/α)(8)-barrel with an additional subdomain built around the C-terminal half of the barrel. The structures revealed high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with structures of other GH17 endo-1,3-β-glucanases revealed differences in the arrangement of the secondary-structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate-binding patterns. The crystal structures revealed an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His(6) tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighbouring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.

  11. Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain*♦

    PubMed Central

    Yokom, Adam L.; Morishima, Yoshihiro; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R.

    2014-01-01

    Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca2+-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis. PMID:24737326

  12. Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms*

    PubMed Central

    Du, Zhihua; Fenn, Sebastian; Tjhen, Richard; James, Thomas L.

    2008-01-01

    Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions. PMID:18701464

  13. Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

    PubMed Central

    Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

    2017-01-01

    Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

  14. Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis.

    PubMed

    Clark, Brian S; Cui, Shuang; Miesfeld, Joel B; Klezovitch, Olga; Vasioukhin, Valeri; Link, Brian A

    2012-05-01

    To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.

  15. Slow dynamics in protein fluctuations revealed by time-structure based independent component analysis: The case of domain motions

    NASA Astrophysics Data System (ADS)

    Naritomi, Yusuke; Fuchigami, Sotaro

    2011-02-01

    Protein dynamics on a long time scale was investigated using all-atom molecular dynamics (MD) simulation and time-structure based independent component analysis (tICA). We selected the lysine-, arginine-, ornithine-binding protein (LAO) as a target protein and focused on its domain motions in the open state. A MD simulation of the LAO in explicit water was performed for 600 ns, in which slow and large-amplitude domain motions of the LAO were observed. After extracting domain motions by rigid-body domain analysis, the tICA was applied to the obtained rigid-body trajectory, yielding slow modes of the LAO's domain motions in order of decreasing time scale. The slowest mode detected by the tICA represented not a closure motion described by a largest-amplitude mode determined by the principal component analysis but a twist motion with a time scale of tens of nanoseconds. The slow dynamics of the LAO were well described by only the slowest mode and were characterized by transitions between two basins. The results show that tICA is promising for describing and analyzing slow dynamics of proteins.

  16. Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

    PubMed Central

    Wu, Hong; Zeng, Hong; Dong, Aiping; Li, Fengling; He, Hao; Senisterra, Guillermo; Seitova, Alma; Duan, Shili; Brown, Peter J.; Vedadi, Masoud; Arrowsmith, Cheryl H.; Schapira, Matthieu

    2013-01-01

    Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation. PMID:24367611

  17. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy.

    PubMed

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-05-12

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

  18. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-05-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

  19. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    PubMed Central

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-01-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels. PMID:25963832

  20. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

    PubMed Central

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

  1. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad

    SciTech Connect

    Karakas,E.; Truglio, J.; Croteau, D.; Rhau, B.; Wang, L.; Van Houten, B.; Kisker, C.

    2007-01-01

    Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

  2. Crystal structure of TIR domain of TLR6 reveals novel dimeric interface of TIR-TIR interaction for toll-like receptor signaling pathway.

    PubMed

    Jang, Tae-Ho; Park, Hyun Ho

    2014-09-23

    Toll-like receptors (TLRs) are responsible for recognition of particular pathogens during the innate immune response and cytoplasmic Toll/interleukin-1 receptor (TIR) domain responsible for downstream signaling. TLR6 working with TLR2 can detect bacterial lipoprotein leading signal for nuclear factor-kappaB activation for immune response. To better understand TLR-mediated signaling event in the innate immune system, in this study, we report the first crystal structure of the TIR domain of TLR6 at 2.2Å resolution. Our structure reveals novel homo-dimerization interfaces, which might be a critical for the interaction with TIR-containing adaptor proteins and itself. We also report structural similarities and differences of TLR6 with those of other TIR domains, which may be functionally relevant. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation, substrate, and inhibitor interactions.

    PubMed

    Gladysheva, Inna P; Sazonova, Irina Y; Chowdhry, Shakeel A; Liu, Lin; Turner, Ryan B; Reed, Guy L

    2002-07-26

    Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.

  4. Biochemical Characterization of Nonamer Binding Domain of RAG1 Reveals its Thymine Preference with Respect to Length and Position

    PubMed Central

    Raveendran, Deepthi; Raghavan, Sathees C.

    2016-01-01

    RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA. PMID:26742581

  5. Targeted molecular dynamics reveals overall common conformational changes upon hybrid domain swing-out in beta3 integrins.

    PubMed

    Provasi, Davide; Murcia, Marta; Coller, Barry S; Filizola, Marta

    2009-11-01

    The beta3 integrin family members alphaIIbeta3 and alphaVbeta3 signal bidirectionally through long-range allosteric changes, including a transition from a bent unliganded-closed low-affinity state to an extended liganded-open high-affinity state. To obtain an atomic-level description of this transition in an explicit solvent, we carried out targeted molecular dynamics simulations of the headpieces of alphaIIbeta3 and alphaVbeta3 integrins. Although minor differences were observed between these receptors, our results suggest a common transition pathway in which the hybrid domain swing-out is accompanied by conformational changes within the beta3 betaA (I-like) domain that propagate through the alpha7 helix C-terminus, and are followed by the alpha7 helix downward motion and the opening of the beta6-alpha7 loop. Breaking of contact interactions between the beta6-alpha7 loop and the alpha1 helix N-terminus results in helix straightening, internal rearrangements of the specificity determining loop (SDL), movement of the beta1-alpha1 loop toward the metal ion dependent adhesion site (MIDAS), and final changes at the interfaces between the beta3 betaA (I-like) domain and either the hybrid or the alpha beta-propeller domains. Taken together, our results suggest novel testable hypotheses of intradomain and interdomain interactions responsible for beta3 integrin activation.

  6. Metagenomic Analysis of Upwelling-Affected Brazilian Coastal Seawater Reveals Sequence Domains of Type I PKS and Modular NRPS.

    PubMed

    Cuadrat, Rafael R C; Cury, Juliano C; Dávila, Alberto M R

    2015-11-27

    Marine environments harbor a wide range of microorganisms from the three domains of life. These microorganisms have great potential to enable discovery of new enzymes and bioactive compounds for industrial use. However, only ~1% of microorganisms from the environment can currently be identified through cultured isolates, limiting the discovery of new compounds. To overcome this limitation, a metagenomics approach has been widely adopted for biodiversity studies on samples from marine environments. In this study, we screened metagenomes in order to estimate the potential for new natural compound synthesis mediated by diversity in the Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) genes. The samples were collected from the Praia dos Anjos (Angel's Beach) surface water-Arraial do Cabo (Rio de Janeiro state, Brazil), an environment affected by upwelling. In order to evaluate the potential for screening natural products in Arraial do Cabo samples, we used KS (keto-synthase) and C (condensation) domains (from PKS and NRPS, respectively) to build Hidden Markov Models (HMM) models. From both samples, a total of 84 KS and 46 C novel domain sequences were obtained, showing the potential of this environment for the discovery of new genes of biotechnological interest. These domains were classified by phylogenetic analysis and this was the first study conducted to screen PKS and NRPS genes in an upwelling affected sample.

  7. Domain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle x-ray scattering.

    PubMed

    Ellis, Jacqueline; Gutierrez, Aldo; Barsukov, Igor L; Huang, Wei-Cheng; Grossmann, J Günter; Roberts, Gordon C K

    2009-12-25

    NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the (1)H-(15)N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2',5'-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.

  8. The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

    PubMed

    Liu, Xu; Shepherd, Tyson R; Murray, Ann M; Xu, Zhen; Fuentes, Ernesto J

    2013-03-05

    PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites.

  9. Characterization of Bacterial, Archaeal and Eukaryote Symbionts from Antarctic Sponges Reveals a High Diversity at a Three-Domain Level and a Particular Signature for This Ecosystem.

    PubMed

    Rodríguez-Marconi, Susana; De la Iglesia, Rodrigo; Díez, Beatriz; Fonseca, Cássio A; Hajdu, Eduardo; Trefault, Nicole

    2015-01-01

    Sponge-associated microbial communities include members from the three domains of life. In the case of bacteria, they are diverse, host specific and different from the surrounding seawater. However, little is known about the diversity and specificity of Eukarya and Archaea living in association with marine sponges. This knowledge gap is even greater regarding sponges from regions other than temperate and tropical environments. In Antarctica, marine sponges are abundant and important members of the benthos, structuring the Antarctic marine ecosystem. In this study, we used high throughput ribosomal gene sequencing to investigate the three-domain diversity and community composition from eight different Antarctic sponges. Taxonomic identification reveals that they belong to families Acarnidae, Chalinidae, Hymedesmiidae, Hymeniacidonidae, Leucettidae, Microcionidae, and Myxillidae. Our study indicates that there are different diversity and similarity patterns between bacterial/archaeal and eukaryote microbial symbionts from these Antarctic marine sponges, indicating inherent differences in how organisms from different domains establish symbiotic relationships. In general, when considering diversity indices and number of phyla detected, sponge-associated communities are more diverse than the planktonic communities. We conclude that three-domain microbial communities from Antarctic sponges are different from surrounding planktonic communities, expanding previous observations for Bacteria and including the Antarctic environment. Furthermore, we reveal differences in the composition of the sponge associated bacterial assemblages between Antarctic and tropical-temperate environments and the presence of a highly complex microbial eukaryote community, suggesting a particular signature for Antarctic sponges, different to that reported from other ecosystems.

  10. The Structure of the Tiam1 PDZ Domain/Phospho-Syndecan1 Complex Reveals a Ligand Conformation that Modulates Protein Dynamics

    PubMed Central

    Liu, Xu; Shepherd, Tyson R.; Murray, Ann M.; Xu, Zhen; Fuentes, Ernesto J.

    2014-01-01

    SUMMARY PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites. PMID:23395182

  11. Structure of Human J-type Co-chaperone HscB Reveals a Tetracysteine Metal-binding Domain*S⃞

    PubMed Central

    Bitto, Eduard; Bingman, Craig A.; Bittova, Lenka; Kondrashov, Dmitry A.; Bannen, Ryan M.; Fox, Brian G.; Markley, John L.; Phillips, George N.

    2008-01-01

    Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0Å crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX9–13FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals. PMID:18713742

  12. NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1.

    PubMed

    Wang, Wei; Lim, Liangzhong; Baskaran, Yohendran; Manser, Ed; Song, Jianxing

    2013-08-16

    Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Characterization of Bacterial, Archaeal and Eukaryote Symbionts from Antarctic Sponges Reveals a High Diversity at a Three-Domain Level and a Particular Signature for This Ecosystem

    PubMed Central

    Rodríguez-Marconi, Susana; De la Iglesia, Rodrigo; Díez, Beatriz; Fonseca, Cássio A.; Hajdu, Eduardo; Trefault, Nicole

    2015-01-01

    Sponge-associated microbial communities include members from the three domains of life. In the case of bacteria, they are diverse, host specific and different from the surrounding seawater. However, little is known about the diversity and specificity of Eukarya and Archaea living in association with marine sponges. This knowledge gap is even greater regarding sponges from regions other than temperate and tropical environments. In Antarctica, marine sponges are abundant and important members of the benthos, structuring the Antarctic marine ecosystem. In this study, we used high throughput ribosomal gene sequencing to investigate the three-domain diversity and community composition from eight different Antarctic sponges. Taxonomic identification reveals that they belong to families Acarnidae, Chalinidae, Hymedesmiidae, Hymeniacidonidae, Leucettidae, Microcionidae, and Myxillidae. Our study indicates that there are different diversity and similarity patterns between bacterial/archaeal and eukaryote microbial symbionts from these Antarctic marine sponges, indicating inherent differences in how organisms from different domains establish symbiotic relationships. In general, when considering diversity indices and number of phyla detected, sponge-associated communities are more diverse than the planktonic communities. We conclude that three-domain microbial communities from Antarctic sponges are different from surrounding planktonic communities, expanding previous observations for Bacteria and including the Antarctic environment. Furthermore, we reveal differences in the composition of the sponge associated bacterial assemblages between Antarctic and tropical-temperate environments and the presence of a highly complex microbial eukaryote community, suggesting a particular signature for Antarctic sponges, different to that reported from other ecosystems. PMID:26421612

  14. Intrinsic Differences in Backbone Dynamics between Wild Type and DNA-Contact Mutants of the p53 DNA Binding Domain Revealed by Nuclear Magnetic Resonance Spectroscopy.

    PubMed

    Rasquinha, Juhi A; Bej, Aritra; Dutta, Shraboni; Mukherjee, Sujoy

    2017-09-19

    Mutations in p53's DNA binding domain (p53DBD) are associated with 50% of all cancers, making it an essential system to investigate and understand the genesis and progression of cancer. In this work, we studied the changes in the structure and dynamics of wild type p53DBD in comparison with two of its "hot-spot" DNA-contact mutants, R248Q and R273H, by analysis of backbone amide chemical shift perturbations and (15)N spin relaxation measurements. The results of amide chemical shift changes indicated significantly more perturbations in the R273H mutant than in wild type and R248Q p53DBD. Analysis of (15)N spin relaxation rates and the resulting nuclear magnetic resonance order parameters suggests that for most parts, the R248Q mutant exhibits limited conformational flexibility and is similar to the wild type protein. In contrast, R273H showed significant backbone dynamics extending up to its β-sandwich scaffold in addition to motions along the DNA binding interface. Furthermore, comparison of rotational correlation times between the mutants suggests that the R273H mutant, with a higher correlation time, forms an enlarged structural fold in comparison to the R248Q mutant and wild type p53DBD. Finally, we identify three regions in these proteins that show conformational flexibility to varying degrees, which suggests that the R273H mutant, in addition to being a DNA-contact mutation, exhibits properties of a conformational mutant.

  15. Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane.

    PubMed

    Legan, P Kevin; Goodyear, Richard J; Morín, Matías; Mencia, Angeles; Pollard, Hilary; Olavarrieta, Leticia; Korchagina, Julia; Modamio-Hoybjor, Silvia; Mayo, Fernando; Moreno, Felipe; Moreno-Pelayo, Miguel-Angel; Richardson, Guy P

    2014-05-15

    Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype-phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the Tecta(L1820F,G1824D/+) mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the Tecta(C1837G/+) mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the Tecta(C1619S/+) mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8-40 kHz range are elevated by 30-40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20-30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

  16. Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

    PubMed

    Mühlbacher, Wolfgang; Mayer, Andreas; Sun, Mai; Remmert, Michael; Cheung, Alan C M; Niesser, Jürgen; Soeding, Johannes; Cramer, Patrick

    2015-10-01

    CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

  17. Structure of the iSH2 domain of Human phosphatidylinositol 3-kinase p85 beta Subunit Reveals Conformational Plasticity in the Interhelical Turn Region

    SciTech Connect

    C Schauder; L Ma; R Krug; G Montelione; R Guan

    2011-12-31

    Phosphatidylinositol 3-kinase (PI3K) proteins actively trigger signaling pathways leading to cell growth, proliferation and survival. These proteins have multiple isoforms and consist of a catalytic p110 subunit and a regulatory p85 subunit. The iSH2 domain of the p85 {beta} isoform has been implicated in the binding of nonstructural protein 1 (NS1) of influenza A viruses. Here, the crystal structure of human p85 {beta} iSH2 determined to 3.3 {angstrom} resolution is reported. The structure reveals that this domain mainly consists of a coiled-coil motif. Comparison with the published structure of the bovine p85 {beta} iSH2 domain bound to the influenza A virus nonstructural protein 1 indicates that little or no structural change occurs upon complex formation. By comparing this human p85 {beta} iSH2 structure with the bovine p85 {beta} iSH2 domain, which shares 99% sequence identity, and by comparing the multiple conformations observed within the asymmetric unit of the bovine iSH2 structure, it was found that this coiled-coil domain exhibits a certain degree of conformational variability or 'plasticity' in the interhelical turn region. It is speculated that this plasticity of p85 {beta} iSH2 may play a role in regulating its functional and molecular-recognition properties.

  18. Structural Context of Disease-Associated Mutations and Putative Mechanism of Autoinhibition Revealed by X-Ray Crystallographic Analysis of the EZH2-SET Domain

    PubMed Central

    Antonysamy, Stephen; Condon, Bradley; Druzina, Zhanna; Bonanno, Jeffrey B.; Gheyi, Tarun; Zhang, Feiyu; MacEwan, Iain; Zhang, Aiping; Ashok, Sheela; Rodgers, Logan; Russell, Marijane; Gately Luz, John

    2013-01-01

    The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain. PMID:24367637

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

    SciTech Connect

    Krejciríková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Rezácová, Pavlína; Brynda, Jirí

    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{sub d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.

  20. Lateral distribution of the transmembrane domain of influenza virus hemagglutinin revealed by time-resolved fluorescence imaging.

    PubMed

    Scolari, Silvia; Engel, Stephanie; Krebs, Nils; Plazzo, Anna Pia; De Almeida, Rodrigo F M; Prieto, Manuel; Veit, Michael; Herrmann, Andreas

    2009-06-05

    Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577-589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

  1. Sub-terahertz frequency-domain spectroscopy reveals single-grain mobility and scatter influence of large-area graphene.

    PubMed

    Cervetti, Christian; Heintze, Eric; Gorshunov, Boris; Zhukova, Elena; Lobanov, Svyatoslav; Hoyer, Alexander; Burghard, Marko; Kern, Klaus; Dressel, Martin; Bogani, Lapo

    2015-04-24

    The response of individual domains in wafer-sized chemical vapor deposition graphene is measured by contactless sub-terahertz interferometry, observing the intrinsic optical conductance and reaching very high mobility values. It is shown that charged scatterers lim