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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. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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)

  10. 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.

  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. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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 =…

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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…

  2. The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

    PubMed

    Gilbert, Hannah E; Asokan, Rengasamy; Holers, V Michael; Perkins, Stephen J

    2006-10-06

    Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  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. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR.

    PubMed

    Lange, Anja; Ismail, Mouhamad-Baligh; Rivière, Gwladys; Hologne, Maggy; Lacabanne, Denis; Guillière, Florence; Lancelin, Jean-Marc; Krimm, Isabelle; Walker, Olivier

    2012-09-21

    To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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;…

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  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. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response

    PubMed Central

    Dinesh, Dhurvas Chandrasekaran; Kovermann, Michael; Gopalswamy, Mohanraj; Hellmuth, Antje; Calderón Villalobos, Luz Irina A.; Lilie, Hauke; Balbach, Jochen; Abel, Steffen

    2015-01-01

    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. PMID:25918389

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Functional Interactions of the HHCC Domain of Moloney Murine Leukemia Virus Integrase Revealed by Nonoverlapping Complementation and Zinc-Dependent Dimerization

    PubMed Central

    Yang, Fan; Leon, Oscar; Greenfield, Norma J.; Roth, Monica J.

    1999-01-01

    The retroviral integrase (IN) is required for the integration of viral DNA into the host genome. The N terminus of IN contains an HHCC zinc finger-like motif, which is conserved among all retroviruses. To study the function of the HHCC domain of Moloney murine leukemia virus IN, the first N-terminal 105 residues were expressed independently. This HHCC domain protein is found to complement a completely nonoverlapping construct lacking the HHCC domain for strand transfer, 3′ processing and coordinated disintegration reactions, revealing trans interactions among IN domains. The HHCC domain protein binds zinc at a 1:1 ratio and changes its conformation upon binding to zinc. The presence of zinc within the HHCC domain stimulates selective integration processes. Zinc promotes the dimerization of the HHCC domain and protects it from N-ethylmaleimide modification. These studies dissect and define the requirement for the HHCC domain, the exact function of which remains unknown. PMID:9971758

  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. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis thaliana.

    PubMed Central

    van Drunen, C M; Oosterling, R W; Keultjes, G M; Weisbeek, P J; van Driel, R; Smeekens, S C

    1997-01-01

    The Arabidopsis thaliana genome is currently being sequenced, eventually leading towards the unravelling of all potential genes. We wanted to gain more insight into the way this genome might be organized at the ultrastructural level. To this extent we identified matrix attachment regions demarking potential chromatin domains, in a 16 kb region around the plastocyanin gene. The region was cloned and sequenced revealing six genes in addition to the plastocyanin gene. Using an heterologous in vitro nuclear matrix binding assay, to search for evolutionary conserved matrix attachment regions (MARs), we identified three such MARs. These three MARs divide the region into two small chromatin domains of 5 kb, each containing two genes. Comparison of the sequence of the three MARs revealed a degenerated 21 bp sequence that is shared between these MARs and that is not found elsewhere in the region. A similar sequence element is also present in four other MARs of Arabidopsis.Therefore, this sequence may constitute a landmark for the position of MARs in the genome of this plant. In a genomic sequence database of Arabidopsis the 21 bp element is found approximately once every 10 kb. The compactness of the Arabidopsis genome could account for the high incidence of MARs and MRSs we observed. PMID:9380515

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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.

    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.

  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. 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

  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. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  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 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  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. 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

  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. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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 limit the mobility, validating previous theoretical predictions, and sub-terahertz quality assessment is demonstrated, as necessary for large-scale applications in touchscreens, as well as wearable and optoelectronic devices.

  19. In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

    RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.

  20. In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference

    NASA Astrophysics Data System (ADS)

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

    RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.

  1. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    NASA Astrophysics Data System (ADS)

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-02-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

  2. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    PubMed Central

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-01-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes. PMID:26861908

  3. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis

    SciTech Connect

    Baird, Nathan J.; Ferré-D'Amaré, Adrian R.

    2010-05-25

    Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.

  4. Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains.

    PubMed

    Van der Aa, Niels; Cheng, Jiqiu; Mateiu, Ligia; Zamani Esteki, Masoud; Kumar, Parveen; Dimitriadou, Eftychia; Vanneste, Evelyne; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2013-04-01

    Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell's copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

  5. Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis

    PubMed Central

    Daskalov, Asen; Habenstein, Birgit; Sabaté, Raimon; Berbon, Mélanie; Martinez, Denis; Chaignepain, Stéphane; Coulary-Salin, Bénédicte; Hofmann, Kay; Loquet, Antoine; Saupe, Sven J.

    2016-01-01

    Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis. PMID:26903619

  6. Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis.

    PubMed

    Daskalov, Asen; Habenstein, Birgit; Sabaté, Raimon; Berbon, Mélanie; Martinez, Denis; Chaignepain, Stéphane; Coulary-Salin, Bénédicte; Hofmann, Kay; Loquet, Antoine; Saupe, Sven J

    2016-03-08

    Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.

  7. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes1[OPEN

    PubMed Central

    Šantrůček, Jiří; Vukašinović, Nemanja

    2017-01-01

    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. PMID:28082718

  8. Human-Mouse Chimeras With Normal Expression and Function Reveal That Major Domain Swapping is Tolerated by P-glycoprotein (ABCB1)

    PubMed Central

    Pluchino, Kristen M.; Hall, Matthew D.; Moen, Janna K.; Chufan, Eduardo E.; Fetsch, Patricia A.; Shukla, Suneet; Gill, Deborah R.; Hyde, Stephen C.; Xia, Di; Ambudkar, Suresh V.; Gottesman, Michael M.

    2017-01-01

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-crosslinking with [125I]-Iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug-binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes. PMID:26820614

  9. Structural investigations of the p53/p73 homologs from the tunicate species Ciona intestinalis reveal the sequence requirements for the formation of a tetramerization domain.

    PubMed

    Heering, Jan; Jonker, Hendrik R A; Löhr, Frank; Schwalbe, Harald; Dötsch, Volker

    2016-02-01

    Most members of the p53 family of transcription factors form tetramers. Responsible for determining the oligomeric state is a short oligomerization domain consisting of one β-strand and one α-helix. With the exception of human p53 all other family members investigated so far contain a second α-helix as part of their tetramerization domain. Here we have used nuclear magnetic resonance spectroscopy to characterize the oligomerization domains of the two p53-like proteins from the tunicate Ciona intestinalis, representing the closest living relative of vertebrates. Structure determination reveals for one of the two proteins a new type of packing of this second α-helix on the core domain that was not predicted based on the sequence, while the other protein does not form a second helix despite the presence of crucial residues that are conserved in all other family members that form a second helix. By mutational analysis, we identify a proline as well as large hydrophobic residues in the hinge region between both helices as the crucial determinant for the formation of a second helix.

  10. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction.

    PubMed

    Crowe, Brandon L; Larue, Ross C; Yuan, Chunhua; Hess, Sonja; Kvaratskhelia, Mamuka; Foster, Mark P

    2016-02-23

    The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a protein-protein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a well-structured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.

  11. Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding

    SciTech Connect

    Sidote,D.; Barbieri, C.; Wu, T.; Stock, A.

    2008-01-01

    The LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.6 Angstroms resolution. The structure establishes a 10-stranded {beta} fold for the LytTR domain and reveals its mode of interaction with DNA. Residues within loop regions of AgrA contact two successive major grooves and the intervening minor groove on one face of the oligonucleotide duplex, inducing a substantial bend in the DNA. Loss of DNA binding upon substitution of key interacting residues in AgrA supports the observed binding mode. This mode of protein-DNA interaction provides a potential target for future antimicrobial drug design.

  12. Phosphopeptide analysis reveals two discrete clusters of phosphorylation in the N-terminus and the Roc domain of the Parkinson-disease associated protein kinase LRRK2.

    PubMed

    Gloeckner, Christian Johannes; Boldt, Karsten; von Zweydorf, Felix; Helm, Sandra; Wiesent, Ludwig; Sarioglu, Hakan; Ueffing, Marius

    2010-04-05

    Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity associate with familial forms of Parkinson disease (PD). As phosphorylation determines the functional state of most protein kinases, we systematically mapped LRRK2 phosphorylation sites by mass spectrometry. Our analysis revealed a high degree of constitutive phosphorylation in a narrow serine-rich region preceding the LRR-domain. Allowing de novo autophosphorylation of purified LRRK2 in an in vitro autokinase assay prior to mass spectrometric analysis, we discovered multiple sites of autophosphorylation. Solely serine and threonine residues were found phosphorylated suggesting LRRK2 as a true serine threonine kinase. Autophosphorylation mainly targets the ROC GTPase domain and its clustering around the GTP binding pocket of ROC suggests cross-regulatory activity between kinase and Roc domain. In conclusion, the phosphoprotein LRRK2 functions as an autocatalytically active serine threonine kinase. Clustering of phosphosites within two discrete domains suggest that phosphorylation may regulate its biological functions in a yet unknown fashion.

  13. Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements.

    PubMed

    Lundgren, Stina; Andersen, Birgit; Piskur, Jure; Dobritzsch, Doreen

    2007-12-07

    Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

  14. Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane*

    PubMed Central

    Ji, Chen; Zhang, Yongdeng; Xu, Pingyong; Xu, Tao; Lou, Xuelin

    2015-01-01

    Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale. PMID:26396197

  15. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  16. Polymer model with Epigenetic Recoloring Reveals a Pathway for the de novo Establishment and 3D Organization of Chromatin Domains

    NASA Astrophysics Data System (ADS)

    Michieletto, D.; Orlandini, E.; Marenduzzo, D.

    2016-10-01

    One of the most important problems in development is how epigenetic domains can first be established, and then maintained, within cells. To address this question, we propose a framework that couples three-dimensional chromatin folding dynamics to a "recoloring" process modeling the writing of epigenetic marks. Because many intrachromatin interactions are mediated by bridging proteins, we consider a "two-state" model with self-attractive interactions between two epigenetic marks that are alike (either active or inactive). This model displays a first-order-like transition between a swollen, epigenetically disordered phase and a compact, epigenetically coherent chromatin globule. If the self-attraction strength exceeds a threshold, the chromatin dynamics becomes glassy, and the corresponding interaction network freezes. By modifying the epigenetic read-write process according to more biologically inspired assumptions, our polymer model with recoloring recapitulates the ultrasensitive response of epigenetic switches to perturbations and accounts for long-lived multidomain conformations, strikingly similar to the topologically associating domains observed in eukaryotic chromosomes.

  17. Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis

    PubMed Central

    Neems, Daniel S.; Garza-Gongora, Arturo G.; Smith, Erica D.; Kosak, Steven T.

    2016-01-01

    The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation. PMID:26957603

  18. The FasFADD death domain complex structure reveals the basis of DISC assembly and disease mutations

    SciTech Connect

    Wang, Liwei; Yang, Jin Kuk; Kabaleeswaran, Venkataraman; Rice, Amanda J.; Cruz, Anthony C.; Park, Ah Young; Yin, Qian; Damko, Ermelinda; Jang, Se Bok; Raunser, Stefan; Robinson, Carol V.; Siegel, Richard M.; Walz, Thomas; Wu, Hao

    2010-10-10

    The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

  19. Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates

    PubMed Central

    Turner, Kendrick B.; Naciri, Jennifer; Liu, Jinny L.; Anderson, George P.; Goldman, Ellen R.; Zabetakis, Dan

    2016-01-01

    Next-Generation Sequencing and bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library. In this work, we constructed a cDNA library of single domain antibodies from a llama immunized with staphylococcal enterotoxin B. The resulting library was sequenced, resulting in approximately 8.5 million sequences with 5.4 million representing intact, useful sequences. The sequenced library was interrogated using sequences of known SEB-binding single domain antibodies from the library obtained through phage display panning methods in a previous study. New antibodies were identified, produced, and characterized, and were shown to have affinities and melting temperatures comparable to those obtained by traditional panning methods. This demonstrates the utility of using NGS as a complementary tool to phage-displayed biopanning as a means for rapidly obtaining additional antibodies from an immune library. It also shows that phage display, using a library of high diversity, is able to select high quality antibodies even when they are low in frequency. PMID:26895405

  20. Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family.

    PubMed

    Ran, Tingting; Gao, Yanyan; Marsh, May; Zhu, Wenjun; Wang, Meitian; Mao, Xiang; Xu, Langlai; Xu, Dongqing; Wang, Weiwu

    2013-01-01

    Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid-histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid-histidine-water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C-C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

  1. The crystal structure of the D-alanine-D-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding.

    PubMed

    Huynh, Kim-Hung; Hong, Myoung-ki; Lee, Clarice; Tran, Huyen-Thi; Lee, Sang Hee; Ahn, Yeh-Jin; Cha, Sun-Shin; Kang, Lin-Woo

    2015-11-01

    Acinetobacter baumannii, which is emerging as a multidrug-resistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.

  2. A thermodynamic study of the third PDZ domain of MAGUK neuronal protein PSD-95 reveals a complex three-state folding behavior.

    PubMed

    Murciano-Calles, Javier; Martinez, Jose C; Marin-Argany, Marta; Villegas, Sandra; Cobos, Eva S

    2014-01-01

    The relevance of the C-terminal α helix of the PDZ3 domain of PSD95 in its unfolding process has been explored by achieving the thermodynamic characterization of a construct where the sequence of the nine residues corresponding to such motif has been deleted. Calorimetric traces at neutral pH require the application of a three-state model displaying three different equilibrium processes in which the intermediate state self-associates upon heating, being stable and populated in a wide temperature range. Temperature scans followed by circular dichroism, Fourier transform infrared spectroscopy and dynamic light scattering support the presence of such oligomeric-partially folded species. This study reveals that the deletion of the α3-helix sequence results in a more complex description of the domain unfolding.

  3. The Structure of the Dead ringer-DNA complex reveals how AT-rich interaction domains (ARIDs) recognize DNA

    SciTech Connect

    Iwahara, Junji; Iwahara, Mizuho; Daughdrill, Gary W.; Ford, Joe J.; Clubb, Robert T.

    2002-03-01

    The AT-rich interaction domain (ARID) is a DNA-binding module found in many eukaryotic transcription factors. Using NMR Spectroscopy, we have determined the first ever three-dimensional structure of an ARID-DNA complex (mol.wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster, ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non-canonical helix-turn-helix motif, and through a concomitant structural rearrangement. that produces stabilizing contacts from a B-hairpin. Dead ringer's preference for a AT-rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine thymine base step.

  4. Biphasic Hoxd Gene Expression in Shark Paired Fins Reveals an Ancient Origin of the Distal Limb Domain

    PubMed Central

    Freitas, Renata; Zhang, GuangJun; Cohn, Martin J.

    2007-01-01

    The evolutionary transition of fins to limbs involved development of a new suite of distal skeletal structures, the digits. During tetrapod limb development, genes at the 5′ end of the HoxD cluster are expressed in two spatiotemporally distinct phases. In the first phase, Hoxd9-13 are activated sequentially and form nested domains along the anteroposterior axis of the limb. This initial phase patterns the limb from its proximal limit to the middle of the forearm. Later in development, a second wave of transcription results in 5′ HoxD gene expression along the distal end of the limb bud, which regulates formation of digits. Studies of zebrafish fins showed that the second phase of Hox expression does not occur, leading to the idea that the origin of digits was driven by addition of the distal Hox expression domain in the earliest tetrapods. Here we test this hypothesis by investigating Hoxd gene expression during paired fin development in the shark Scyliorhinus canicula, a member of the most basal lineage of jawed vertebrates. We report that at early stages, 5′Hoxd genes are expressed in anteroposteriorly nested patterns, consistent with the initial wave of Hoxd transcription in teleost and tetrapod paired appendages. Unexpectedly, a second phase of expression occurs at later stages of shark fin development, in which Hoxd12 and Hoxd13 are re-expressed along the distal margin of the fin buds. This second phase is similar to that observed in tetrapod limbs. The results indicate that a second, distal phase of Hoxd gene expression is not uniquely associated with tetrapod digit development, but is more likely a plesiomorphic condition present the common ancestor of chondrichthyans and osteichthyans. We propose that a temporal extension, rather than de novo activation, of Hoxd expression in the distal part of the fin may have led to the evolution of digits. PMID:17710153

  5. Local domains of motor cortical activity revealed by fiber-optic calcium recordings in behaving nonhuman primates

    PubMed Central

    Adelsberger, Helmuth; Zainos, Antonio; Alvarez, Manuel; Romo, Ranulfo; Konnerth, Arthur

    2014-01-01

    Brain mapping experiments involving electrical microstimulation indicate that the primary motor cortex (M1) directly regulates muscle contraction and thereby controls specific movements. Possibly, M1 contains a small circuit “map” of the body that is formed by discrete local networks that code for specific movements. Alternatively, movements may be controlled by distributed, larger-scale overlapping circuits. Because of technical limitations, it remained unclear how movement-determining circuits are organized in M1. Here we introduce a method that allows the functional mapping of small local neuronal circuits in awake behaving nonhuman primates. For this purpose, we combined optic-fiber–based calcium recordings of neuronal activity and cortical microstimulation. The method requires targeted bulk loading of synthetic calcium indicators (e.g., OGB-1 AM) for the staining of neuronal microdomains. The tip of a thin (200 µm) optical fiber can detect the coherent activity of a small cluster of neurons, but is insensitive to the asynchronous activity of individual cells. By combining such optical recordings with microstimulation at two well-separated sites of M1, we demonstrate that local cortical activity was tightly associated with distinct and stereotypical simple movements. Increasing stimulation intensity increased both the amplitude of the movements and the level of neuronal activity. Importantly, the activity remained local, without invading the recording domain of the second optical fiber. Furthermore, there was clear response specificity at the two recording sites in a trained behavioral task. Thus, the results provide support for movement control in M1 by local neuronal clusters that are organized in discrete cortical domains. PMID:24344287

  6. Local domains of motor cortical activity revealed by fiber-optic calcium recordings in behaving nonhuman primates.

    PubMed

    Adelsberger, Helmuth; Zainos, Antonio; Alvarez, Manuel; Romo, Ranulfo; Konnerth, Arthur

    2014-01-07

    Brain mapping experiments involving electrical microstimulation indicate that the primary motor cortex (M1) directly regulates muscle contraction and thereby controls specific movements. Possibly, M1 contains a small circuit "map" of the body that is formed by discrete local networks that code for specific movements. Alternatively, movements may be controlled by distributed, larger-scale overlapping circuits. Because of technical limitations, it remained unclear how movement-determining circuits are organized in M1. Here we introduce a method that allows the functional mapping of small local neuronal circuits in awake behaving nonhuman primates. For this purpose, we combined optic-fiber-based calcium recordings of neuronal activity and cortical microstimulation. The method requires targeted bulk loading of synthetic calcium indicators (e.g., OGB-1 AM) for the staining of neuronal microdomains. The tip of a thin (200 µm) optical fiber can detect the coherent activity of a small cluster of neurons, but is insensitive to the asynchronous activity of individual cells. By combining such optical recordings with microstimulation at two well-separated sites of M1, we demonstrate that local cortical activity was tightly associated with distinct and stereotypical simple movements. Increasing stimulation intensity increased both the amplitude of the movements and the level of neuronal activity. Importantly, the activity remained local, without invading the recording domain of the second optical fiber. Furthermore, there was clear response specificity at the two recording sites in a trained behavioral task. Thus, the results provide support for movement control in M1 by local neuronal clusters that are organized in discrete cortical domains.

  7. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    PubMed Central

    Stroehlein, Andreas J.; Young, Neil D.; Korhonen, Pasi K.; Chang, Bill C. H.; Sternberg, Paul W.; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B.

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  8. Fast Helix Formation in the B Domain of Protein A Revealed by Site-Specific Infrared Probes

    PubMed Central

    Davis, Caitlin M.; Cooper, A. Kat; Dyer, R. Brian

    2015-01-01

    Comparison of experimental and computational protein folding studies can be difficult because of differences in structural resolution. Isotope-edited infrared spectroscopy offers a direct measure of structural changes involved in protein folding at the single-residue level. Here we demonstrate the increased resolution of site-specific infrared probes to the peptide backbone in the B domain of staphylococcal protein A (BdpA). 13C=18O-labeled methionine was incorporated into each of the helices using recombinant protein expression. Laser-induced temperature jumps coupled with infrared spectroscopy were used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation kinetics of the buried helices, solvated helices, and labeled positions were measured independently by probing the corresponding bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe a fast nanosecond phase and slower microsecond phase at each position. We find at least partial formation of helices 1–3 in the fast intermediate state that precedes the transition state. These measurements provide direct, time-resolved experimental evidence of the early formation of partial helical structure in helices 1 and 3, supporting folding models proposed by computer simulations. PMID:25706439

  9. Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

    SciTech Connect

    Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.; Deisenhofer, Johann; Goldstein, Joseph L.; Brown, Michael S.; Infante, Rodney E.

    2010-09-21

    LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

  10. Looking, seeing and believing in autism: Eye movements reveal how subtle cognitive processing differences impact in the social domain.

    PubMed

    Benson, Valerie; Castelhano, Monica S; Howard, Philippa L; Latif, Nida; Rayner, Keith

    2016-08-01

    Adults with High Functioning Autism Spectrum Disorders (ASD) viewed scenes with people in them, while having their eye movements recorded. The task was to indicate, using a button press, whether the pictures were normal, or in some way weird or odd. Oddities in the pictures were categorized as violations of either perceptual or social norms. Compared to a Typically Developed (TD) control group, the ASD participants were equally able to categorize the scenes as odd or normal, but they took longer to respond. The eye movement patterns showed that the ASD group made more fixations and revisits to the target areas in the odd scenes compared with the TD group. Additionally, when the ASD group first fixated the target areas in the scenes, they failed to initially detect the social oddities. These two findings have clear implications for processing difficulties in ASD for the social domain, where it is important to detect social cues on-line, and where there is little opportunity to go back and recheck possible cues in fast dynamic interactions. Autism Res 2016, 9: 879-887. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

  11. Fast helix formation in the B domain of protein A revealed by site-specific infrared probes.

    PubMed

    Davis, Caitlin M; Cooper, A Kat; Dyer, R Brian

    2015-03-10

    Comparison of experimental and computational protein folding studies can be difficult because of differences in structural resolution. Isotope-edited infrared spectroscopy offers a direct measure of structural changes involved in protein folding at the single-residue level. Here we demonstrate the increased resolution of site-specific infrared probes to the peptide backbone in the B domain of staphylococcal protein A (BdpA). (13)C═(18)O-labeled methionine was incorporated into each of the helices using recombinant protein expression. Laser-induced temperature jumps coupled with infrared spectroscopy were used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation kinetics of the buried helices, solvated helices, and labeled positions were measured independently by probing the corresponding bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe a fast nanosecond phase and slower microsecond phase at each position. We find at least partial formation of helices 1-3 in the fast intermediate state that precedes the transition state. These measurements provide direct, time-resolved experimental evidence of the early formation of partial helical structure in helices 1 and 3, supporting folding models proposed by computer simulations.

  12. Exchanges of genomic domains between poliovirus and other cocirculating species C enteroviruses reveal a high degree of plasticity

    PubMed Central

    Bessaud, Maël; Joffret, Marie-Line; Blondel, Bruno; Delpeyroux, Francis

    2016-01-01

    The attenuated Sabin strains contained in the oral poliomyelitis vaccine are genetically unstable, and their circulation in poorly immunized populations can lead to the emergence of pathogenic circulating vaccine-derived polioviruses (cVDPVs). The recombinant nature of most cVDPV genomes and the preferential presence of genomic sequences from certain cocirculating non-polio enteroviruses of species C (EV-Cs) raise questions about the permissiveness of genetic exchanges between EV-Cs and the phenotypic impact of such exchanges. We investigated whether functional constraints limited genetic exchanges between Sabin strains and other EV-Cs. We bypassed the natural recombination events by constructing 29 genomes containing a Sabin 2 capsid-encoding sequence and other sequences from Sabin 2 or from non-polio EV-Cs. Most genomes were functional. All recombinant viruses replicated similarly in vitro, but recombination modulated plaque size and temperature sensitivity. All viruses with a 5′UTR from Sabin 2 were attenuated in mice, whereas almost all viruses with a non-polio 5′UTR caused disease. These data highlight the striking conservation of functional compatibility between different genetic domains of cocirculating EV-Cs. This aspect is only one of the requirements for the generation of recombinant cVDPVs in natural conditions, but it may facilitate the generation of viable intertypic recombinants with diverse phenotypic features, including pathogenicity. PMID:27958320

  13. Hinge bending within the cytokine receptor superfamily revealed by the 2.4 A crystal structure of the extracellular domain of rabbit tissue factor.

    PubMed Central

    Muller, Y. A.; Kelley, R. F.; de Vos, A. M.

    1998-01-01

    Tissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF x FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 A resolution and refined to a crystallographic R-value of 19.1% (free R-value, 27.7%). Like the human homologue, the extracellular portion consists of two fibronectin type III domains connected by a short alpha-helical segment. Unexpectedly, the two molecules in the crystallographic asymmetric unit differ in their relative domain-domain orientation, revealing unsuspected hinge motion consisting of a rotation of about 12.7 degrees around an axis intersecting the linker segment at residue 106. Superposition of rabbit tissue factor with free and bound human tissue factor allows for the detection of an identical, albeit smaller, hinge motion in human TF induced upon binding of FVIIa. This raises the possibility that a very similar hinge axis may be present in other members of the cytokine receptor superfamily. PMID:9605315

  14. Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

    SciTech Connect

    Maiti, Tushar K.; Permaul, Michelle; Boudreaux, David A.; Mahanic, Christina; Mauney, Sarah; Das, Chittaranjan

    2012-10-25

    Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.

  15. Biochemical characterization of hyperactive β2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains

    PubMed Central

    Sosa, Maria Soledad; Lewin, Nancy E.; Choi, Sung-Hee; Blumberg, Peter M.; Kazanietz, Marcelo G.

    2009-01-01

    Recent studies established that the Rac-GAP β2-chimaerin plays important roles in development, neuritogenesis, and cancer progression. A unique feature of β2-chimaerin is that it can be activated by phorbol esters and the lipid second messenger diacylglycerol (DAG), which bind with high affinity to its C1 domain and promote β2-chimaerin translocation to membranes, leading to the inactivation of the small G-protein Rac. Crystallographic evidence and cellular studies suggest that β2-chimaerin remains in an inactive conformation in the cytosol with the C1 domain inaccessible to ligands. We developed a series of β2-chimaerin point mutants in which intramolecular contacts that occlude the C1 domain have been disrupted. These mutants showed enhanced translocation in response to phorbol 12-myristate 13-acetate (PMA) in cells. Binding assays using [3H]phorbol 12, 13-dibutyrate ([3H]PDBu) revealed that internal contact mutants have a reduced acidic phospholipid requirement for phorbol ester binding. Moreover, disruption of intramolecular contacts enhances binding of β2-chimaerin to acidic phospholipid vesicles and confers enhanced Rac-GAP activity in vitro. These studies suggest that β2-chimaerin must undergo a conformational rearrangement in order to expose its lipid binding sites and become activated. PMID:19618918

  16. An ion selectivity filter in the extracellular domain of Cys-loop receptors reveals determinants for ion conductance.

    PubMed

    Hansen, Scott B; Wang, Hai-Long; Taylor, Palmer; Sine, Steven M

    2008-12-26

    Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

  17. Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding*

    PubMed Central

    Álvaro-Benito, Miguel; Polo, Aitana; González, Beatriz; Fernández-Lobato, María; Sanz-Aparicio, Julia

    2010-01-01

    Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases β-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 Å resolution and its complex with fructose at 1.9 Å resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the β-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme. PMID:20181943

  18. A study of the YopD-lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.

    PubMed

    Francis, M S; Aili, M; Wiklund, M L; Wolf-Watz, H

    2000-10-01

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  19. Structures of Ca(V) Ca**2+/CaM-IQ Domain Complexes Reveal Binding Modes That Underlie Calcium-Dependent Inactivation And Facilitation

    SciTech Connect

    Kim, E.Y.; Rumpf, C.H.; Fujiwara, Y.; Cooley, E.S.; Petegem, F.Van; Minor, D.L., Jr.

    2009-05-20

    Calcium influx drives two opposing voltage-activated calcium channel (Ca{sub V}) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca{sup 2+}/calmodulin (Ca{sup 2+}/CaM) lobes produce CDI and CDF through interactions with the Ca{sub V}{alpha}{sub 1} subunit IQ domain. Curiously, Ca{sup 2+}/CaM lobe modulation polarity appears inverted between Ca{sub V}1s and Ca{sub V}2s. Here, we present crystal structures of Ca{sub V}2.1, Ca{sub V}2.2, and Ca{sub V}2.3 Ca{sup 2+}/CaM-IQ domain complexes. All display binding orientations opposite to Ca{sub V}1.2 with a physical reversal of the CaM lobe positions relative to the IQ {alpha}-helix. Titration calorimetry reveals lobe competition for a high-affinity site common to Ca{sub V}1 and Ca{sub V}2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca{sub V}2 Ca{sup 2+}/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca{sub V} feedback modulation and indicate that Ca{sub V}1 and Ca{sub V}2 IQ domains bear a dedicated CDF site that exchanges Ca{sup 2+}/CaM lobe occupants.

  20. Flexible Ablators

    NASA Technical Reports Server (NTRS)

    Stackpoole, Margaret M. (Inventor); Ghandehari, Ehson M. (Inventor); Thornton, Jeremy J. (Inventor); Covington, Melmoth Alan (Inventor)

    2017-01-01

    A low-density article comprising a flexible substrate and a pyrolizable material impregnated therein, methods of preparing, and devices using the article are disclosed. The pyrolizable material pyrolizes above 350 C and does not flow at temperatures below the pyrolysis temperature. The low-density article remains flexible after impregnation and continues to remain flexible when the pyrolizable material is fully pyrolized.

  1. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    SciTech Connect

    Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul; Bae, Kwang-Hee; Park, Jae-Yong; Choi, Wan Sung; Yoon, Ho Sup; Park, Sung Goo; Park, Byoung Chul; Yi, Gwan-Su; Chi, Seung-Wook

    2011-05-20

    Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  2. Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

    SciTech Connect

    Tu, Chao; Tan, Yu-Hong; Shaw, Gary; Zhou, Zheng; Bai, Yawen; Luo, Ray; Ji, Xinhua

    2008-05-01

    The impact of hotspot mutation R282Q on the structure of human p53 DNA-binding domain has been characterized by X-ray crystallography and molecular-dynamics simulations. Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53.

  3. Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap

    NASA Astrophysics Data System (ADS)

    Gu, Shuang; Sushko, Oleksandr; Deery, Evelyne; Warren, Martin J.; Pickersgill, Richard W.

    2015-11-01

    CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel β-sheets have swapped their last β-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold. The trapped ternary complex with coenzyme and product reveals five conserved basic residues that bind the carboxylates of the tetrapyrrole tightly anchoring the product. A loop, disordered in both the apoenzyme and holoenzyme structures, closes around the product further tightening binding. The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK. The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.

  4. Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

    PubMed Central

    Coric, Pascale; Saribas, A. Sami; Abou-Gharbia, Magid; Childers, Wayne; White, Martyn K.

    2014-01-01

    highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic α-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the α-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection. PMID:24672035

  5. Virtual screening on an α-helix to β-strand switchable region of the FGFR2 extracellular domain revealed positive and negative modulators.

    PubMed

    Diaz, Constantino; Corentin, Herbert; Thierry, Vermat; Chantal, Alcouffe; Tanguy, Bozec; David, Sibrac; Jean-Marc, Herbert; Pascual, Ferrara; Françoise, Bono; Edgardo, Ferran

    2014-11-01

    The secondary structure of some protein segments may vary between α-helix and β-strand. To predict these switchable segments, we have developed an algorithm, Switch-P, based solely on the protein sequence. This algorithm was used on the extracellular parts of FGF receptors. For FGFR2, it predicted that β4 and β5 strands of the third Ig-like domain were highly switchable. These two strands possess a high number of somatic mutations associated with cancer. Analysis of PDB structures of FGF receptors confirmed the switchability prediction for β5. We thus evaluated if compound-driven α-helix/β-strand switching of β5 could modulate FGFR2 signaling. We performed the virtual screening of a library containing 1.4 million of chemical compounds with two models of the third Ig-like domain of FGFR2 showing different secondary structures for β5, and we selected 32 compounds. Experimental testing using proliferation assays with FGF7-stimulated SNU-16 cells and a FGFR2-dependent Erk1/2 phosphorylation assay with FGFR2-transfected L6 cells, revealed activators and inhibitors of FGFR2. Our method for the identification of switchable proteinic regions, associated with our virtual screening approach, provides an opportunity to discover new generation of drugs with under-explored mechanism of action.

  6. Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains

    PubMed Central

    Liu, Junfeng; Huang, Jinguang; Zhao, Yanxiang; Liu, Huaian; Wang, Dawei; Yang, Jun; Zhao, Wensheng; Taylor, Ian A.; Peng, You-Liang

    2015-01-01

    The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains. PMID:25550425

  7. 19F NMR Reveals Multiple Conformations at the Dimer Interface of the Non-Structural Protein 1 Effector Domain from Influenza A Virus

    PubMed Central

    Ma, Li-Chung; Swapna, G. V. T.; Leonard, Paul G.; Ladbury, John E.; Krug, Robert M.; Montelione, Gaetano T.

    2014-01-01

    SUMMARY Non-structural protein 1 of influenza A virus, NS1A, is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain (RBD) and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply 19F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan (5-F-Trp), and demonstrate that the 19F signal of Trp187 is a sensitive, direct monitor of the ED helix-helix dimer interface. 19F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein-protein interface, whose rate is over three orders of magnitude faster than the kinetics of ED dimerization. 19F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1Aat concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. PMID:24582435

  8. Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence

    PubMed Central

    de Almeida, Rodrigo F. M.; Borst, JanWillem; Fedorov, Alexander; Prieto, Manuel; Visser, Antonie J. W. G.

    2007-01-01

    The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (lo)/liquid-disordered (ld) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of lo raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the lo phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed. PMID:17449668

  9. Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

    PubMed Central

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

    2017-01-01

    The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

  10. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    PubMed

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  11. Is Spectral-Domain Optical Coherence Tomography Essential for Flexible Treatment Regimens with Ranibizumab for Neovascular Age-Related Macular Degeneration?

    PubMed Central

    Ozkaya, Abdullah; Agca, Alper; Ozgurhan, Engin Bilge; Karakucuk, Yalcin; Yazici, Ahmet Taylan; Demirok, Ahmet

    2013-01-01

    Purpose. To evaluate the ability of spectral-domain optical coherence tomography to detect subtle amounts of retinal fluid when the choroidal neovascularization is detected as inactive via time-domain optical coherence tomography and clinical examination in neovascular age-related macular degeneration (nAMD) patients. Methods. Forty-nine eyes of 49 patients with nAMD after ranibizumab treatment were included in this cross-sectional, prospective study. All patients were imaged with TD-OCT and SD-OCT at the same visit one month after a ranibizumab injection. The presence of subretinal, intraretinal, and subretinal pigment epithelium fluid (subRPE) in SD-OCT was evaluated; also mean central retinal thickness (CRT) and the rate of vitreoretinal surface disorders detected via the two devices were evaluated. Results. The mean CRT via TD-OCT and SD-OCT was 218.1 ± 51.3 and 325.7 ± 78.8 microns. Sixteen patients (32.6%) showed any kind of retinal fluid via SD-OCT. In detail, 8 patients (16.3%) showed subretinal fluid, 10 patients (20.4%) showed intraretinal fluid, and 3 patients (6.1%) showed SubRPE fluid. The ability of detecting vitreoretinal surface disorders was comparable between the two devices, except vitreomacular traction. Conclusion. SD-OCT is essential for the nAMD patients who are on an as-needed treatment regimen with ranibizumab. Only TD-OCT and clinical examination may cause insufficient treatment in this group of patients. PMID:24324880

  12. The structure of Jann_2411 (DUF1470) from Jannaschia sp. at 1.45 Å resolution reveals a new fold (the ABATE domain) and suggests its possible role as a transcription regulator

    PubMed Central

    Bakolitsa, Constantina; Bateman, Alex; Jin, Kevin K.; McMullan, Daniel; Krishna, S. Sri; Miller, Mitchell D.; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Axelrod, Herbert L.; Burra, Prasad; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Elias, Ylva; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Murphy, Kevin D.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry; Trame, Christine B.; Trout, Christina V.; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott; Wilson, Ian A.

    2010-01-01

    The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45 Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators. PMID:20944211

  13. Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

    PubMed Central

    Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

    2010-01-01

    The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

  14. Ketoolivosyl-tetracenomycin C: a new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT

    PubMed Central

    Nybo, S. Eric; Shabaan, Khaled A.; Kharel, Madan K.; Salas, José A.; Méndez, Carmen; Sutardjo, Happy; Rohr, Jürgen

    2012-01-01

    A new tetracenomycin analogue, 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a “sugar plasmid” (pKOL) directing the biosynthesis of NDP-4-keto-l-olivose. This strain harbors all of the genes necessary for production of 8-demethylt-etracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-l-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways. PMID:22361136

  15. X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions*

    PubMed Central

    Chen, Yafang; Savinov, Sergey N.; Mielech, Anna M.; Cao, Thu; Baker, Susan C.; Mesecar, Andrew D.

    2015-01-01

    Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication. PMID:26296883

  16. Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

    PubMed Central

    2010-01-01

    Background Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility. Results We molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins. Conclusion Our findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control. PMID:20170486

  17. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    SciTech Connect

    Carmen Herranz, Ma; Mingarro, Ismael; Pallas, Vicente . E-mail: vpallas@ibmcp.upv.es

    2005-08-15

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

  18. The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1.

    PubMed

    Bhella, David; Goodfellow, Ian G

    2011-11-01

    Caliciviridae are small icosahedral positive-sense RNA-containing viruses and include the human noroviruses, a leading cause of infectious acute gastroenteritis and feline calicivirus (FCV), which causes respiratory illness and stomatitis in cats. FCV attachment and entry is mediated by feline junctional adhesion molecule A (fJAM-A), which binds to the outer face of the capsomere, inducing a conformational change in the capsid that may be important for viral uncoating. Here we present the results of our structural investigation of the virus-receptor interaction and ensuing conformational changes. Cryo-electron microscopy and three-dimensional image reconstruction were used to solve the structure of the virus decorated with a soluble fragment of the receptor at subnanometer resolution. In initial reconstructions, the P domains of the capsid protein VP1 and fJAM-A were poorly resolved. Sorting experiments led to improved reconstructions of the FCV-fJAM-A complex both before and after the induced conformational change, as well as in three transition states. These data showed that the P domain becomes flexible following fJAM-A binding, leading to a loss of icosahedral symmetry. Furthermore, two distinct conformational changes were seen; an anticlockwise rotation of up to 15° of the P domain was observed in the AB dimers, while tilting of the P domain away from the icosahedral 2-fold axis was seen in the CC dimers. A list of putative contact residues was calculated by fitting high-resolution coordinates for fJAM-A and VP1 to the reconstructed density maps, highlighting regions in both virus and receptor important for virus attachment and entry.

  19. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  20. Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion.

    PubMed

    Bose, Sayantan; Welch, Brett D; Kors, Christopher A; Yuan, Ping; Jardetzky, Theodore S; Lamb, Robert A

    2011-12-01

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  1. NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping.

    PubMed

    López-Alonso, Jorge Pedro; Bruix, Marta; Font, Josep; Ribó, Marc; Vilanova, Maria; Jiménez, María Angeles; Santoro, Jorge; González, Carlos; Laurents, Douglas V

    2010-02-10

    Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.

  2. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    PubMed

    Li, Yan; Burclaff, Joseph; Anderson, James T

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  3. Flexible Straws.

    ERIC Educational Resources Information Center

    Prentice, Gerard

    1989-01-01

    Discusses the use of flexible straws for teaching properties of figures and families of shapes. Describes a way to make various two- or three-dimensional geometric shapes. Lists eight advantages of the method. (YP)

  4. Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation

    PubMed Central

    Cheng, Shaohong; Xing, Weirong; Pourteymoor, Sheila; Schulte, Jan

    2016-01-01

    The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice. PMID:26562260

  5. Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation.

    PubMed

    Cheng, Shaohong; Xing, Weirong; Pourteymoor, Sheila; Schulte, Jan; Mohan, Subburaman

    2016-01-01

    The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

  6. Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability

    SciTech Connect

    Hansman, Grant S.; Biertümpfel, Christian; Georgiev, Ivelin; McLellan, Jason S.; Chen, Lei; Zhou, Tongqing; Katayama, Kazuhiko; Kwong, Peter D.

    2011-10-10

    Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal {alpha}fucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical {alpha}fucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.

  7. Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

    PubMed

    Yoshida, Keita; Hozumi, Akiko; Treen, Nicholas; Sakuma, Tetsushi; Yamamoto, Takashi; Shirae-Kurabayashi, Maki; Sasakura, Yasunori

    2017-03-15

    The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.

  8. Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR.

    PubMed

    Wadhwani, Parvesh; Strandberg, Erik; Heidenreich, Nico; Bürck, Jochen; Fanghänel, Susanne; Ulrich, Anne S

    2012-04-18

    The cationic peptide [KIGAKI](3) was designed as an amphiphilic β-strand and serves as a model for β-sheet aggregation in membranes. Here, we have characterized its molecular conformation, membrane alignment, and dynamic behavior using solid-state (19)F NMR. A detailed structure analysis of selectively (19)F-labeled peptides was carried out in oriented DMPC bilayers. It showed a concentration-dependent transition from monomeric β-strands to oligomeric β-sheets. In both states, the rigid (19)F-labeled side chains project straight into the lipid bilayer but they experience very different mobilities. At low peptide-to-lipid ratios ≤1:400, monomeric [KIGAKI](3) swims around freely on the membrane surface and undergoes considerable motional averaging, with essentially uncoupled φ/ψ torsion angles. The flexibility of the peptide backbone in this 2D plane is reminiscent of intrinsically unstructured proteins in 3D. At high concentrations, [KIGAKI](3) self-assembles into immobilized β-sheets, which are untwisted and lie flat on the membrane surface as amyloid-like fibrils. This is the first time the transition of monomeric β-strands into oligomeric β-sheets has been characterized by solid-state NMR in lipid bilayers. It promises to be a valuable approach for studying membrane-induced amyloid formation of many other, clinically relevant peptide systems.

  9. Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

    2011-01-01

    Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

  10. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature.

    PubMed

    El-Guizani, Taissir; Guibert, Clotilde; Triki, Saida; St-Pierre, Benoit; Ducos, Eric

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes.

  11. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface.

    PubMed

    Atkison, James H; Parnham, Stuart; Marcotte, William R; Olsen, Shaun K

    2016-09-02

    Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct.

  12. Complex Regulation by Apetala2 Domain-Containing Transcription Factors Revealed through Analysis of the Stress-Responsive TdCor410b Promoter from Durum Wheat

    PubMed Central

    Eini, Omid; Yang, Nannan; Pyvovarenko, Tatiana; Pillman, Katherine; Bazanova, Natalia; Tikhomirov, Natalia; Eliby, Serik; Shirley, Neil; Sivasankar, Shoba; Tingey, Scott; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2013-01-01

    Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression. PMID:23527011

  13. Analysis of the Costructure of the Simian Virus 40 T-Antigen Origin Binding Domain with Site I Reveals a Correlation between GAGGC Spacing and Spiral Assembly

    PubMed Central

    Meinke, Gretchen; Phelan, Paul J.; Harrison, Celia J.

    2013-01-01

    Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag). The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed. PMID:23269808

  14. The crystal structure of the orphan nuclear receptor NR2E3/PNR ligand binding domain reveals a dimeric auto-repressed conformation.

    PubMed

    Tan, M H Eileen; Zhou, X Edward; Soon, Fen-Fen; Li, Xiaodan; Li, Jun; Yong, Eu-Leong; Melcher, Karsten; Xu, H Eric

    2013-01-01

    Photoreceptor-specific nuclear receptor (PNR, NR2E3) is a key transcriptional regulator of human photoreceptor differentiation and maintenance. Mutations in the NR2E3-encoding gene cause various retinal degenerations, including Enhanced S-cone syndrome, retinitis pigmentosa, and Goldman-Favre disease. Although physiological ligands have not been identified, it is believed that binding of small molecule agonists, receptor desumoylation, and receptor heterodimerization may switch NR2E3 from a transcriptional repressor to an activator. While these features make NR2E3 a potential therapeutic target for the treatment of retinal diseases, there has been a clear lack of structural information for the receptor. Here, we report the crystal structure of the apo NR2E3 ligand binding domain (LBD) at 2.8 Å resolution. Apo NR2E3 functions as transcriptional repressor in cells and the structure of its LBD is in a dimeric auto-repressed conformation. In this conformation, the putative ligand binding pocket is filled with bulky hydrophobic residues and the activation-function-2 (AF2) helix occupies the canonical cofactor binding site. Mutations designed to disrupt either the AF2/cofactor-binding site interface or the dimer interface compromised the transcriptional repressor activity of this receptor. Together, these results reveal several conserved structural features shared by related orphan nuclear receptors, suggest that most disease-causing mutations affect the receptor's structural integrity, and allowed us to model a putative active conformation that can accommodate small ligands in its pocket.

  15. Analysis of the costructure of the simian virus 40 T-antigen origin binding domain with site I reveals a correlation between GAGGC spacing and spiral assembly.

    PubMed

    Meinke, Gretchen; Phelan, Paul J; Harrison, Celia J; Bullock, Peter A

    2013-03-01

    Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag). The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed.

  16. Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding.

    PubMed

    Matsuoka, Eriko; Tanaka, Yoshikazu; Kuroda, Makoto; Shouji, Yuko; Ohta, Toshiko; Tanaka, Isao; Yao, Min

    2011-02-01

    Staphylococci use cell wall-anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall-anchored protein, uro-adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain-truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.

  17. Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding

    PubMed Central

    Matsuoka, Eriko; Tanaka, Yoshikazu; Kuroda, Makoto; Shouji, Yuko; Ohta, Toshiko; Tanaka, Isao; Yao, Min

    2011-01-01

    Staphylococci use cell wall-anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall-anchored protein, uro-adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain-truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus. PMID:21280131

  18. Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination.

    PubMed

    Fukuda, Tomoyuki; Kugou, Kazuto; Sasanuma, Hiroyuki; Shibata, Takehiko; Ohta, Kunihiro

    2008-02-01

    Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.

  19. High muscle mitochondrial volume and aerobic capacity in a small marsupial (Sminthopsis crassicaudata) reveals flexible links between energy-use levels in mammals.

    PubMed

    Dawson, Terence J; Webster, Koa N; Lee, Enhua; Buttemer, William A

    2013-04-01

    comparably sized placentals, with the reverse applying for larger marsupials. The flexibility of energy output in marsupials provides explanations for this pattern. Overall, our data refute widely held notions of mechanistically closely linked relationships between body mass, BMR, FMR and MMR in mammals generally.

  20. SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

    PubMed Central

    2012-01-01

    Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains. PMID:22974441

  1. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    SciTech Connect

    Peng, Shuxia Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  2. Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain.

    PubMed

    Couto, J R; Taylor, M R; Godwin, S G; Ceriani, R L; Peterson, J A

    1996-04-01

    The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

  3. The Crystal Structure of the Orphan Nuclear Receptor NR2E3/PNR Ligand Binding Domain Reveals a Dimeric Auto-Repressed Conformation

    PubMed Central

    Tan, M. H. Eileen; Zhou, X. Edward; Soon, Fen-Fen; Li, Xiaodan; Li, Jun; Yong, Eu-Leong; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Photoreceptor-specific nuclear receptor (PNR, NR2E3) is a key transcriptional regulator of human photoreceptor differentiation and maintenance. Mutations in the NR2E3-encoding gene cause various retinal degenerations, including Enhanced S-cone syndrome, retinitis pigmentosa, and Goldman-Favre disease. Although physiological ligands have not been identified, it is believed that binding of small molecule agonists, receptor desumoylation, and receptor heterodimerization may switch NR2E3 from a transcriptional repressor to an activator. While these features make NR2E3 a potential therapeutic target for the treatment of retinal diseases, there has been a clear lack of structural information for the receptor. Here, we report the crystal structure of the apo NR2E3 ligand binding domain (LBD) at 2.8 Å resolution. Apo NR2E3 functions as transcriptional repressor in cells and the structure of its LBD is in a dimeric auto-repressed conformation. In this conformation, the putative ligand binding pocket is filled with bulky hydrophobic residues and the activation-function-2 (AF2) helix occupies the canonical cofactor binding site. Mutations designed to disrupt either the AF2/cofactor-binding site interface or the dimer interface compromised the transcriptional repressor activity of this receptor. Together, these results reveal several conserved structural features shared by related orphan nuclear receptors, suggest that most disease-causing mutations affect the receptor’s structural integrity, and allowed us to model a putative active conformation that can accommodate small ligands in its pocket. PMID:24069298

  4. Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain

    PubMed Central

    2016-01-01

    The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of 13C-, 2H-, and 15N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event. PMID:25844500

  5. Degradation and adsorption characteristics of PHB depolymerase as revealed by kinetics of mutant enzymes with amino acid substitution in substrate-binding domain.

    PubMed

    Hiraishi, Tomohiro; Komiya, Naoya; Matsumoto, Nobuhiko; Abe, Hideki; Fujita, Masahiro; Maeda, Mizuo

    2010-01-11

    Extracelluar Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ(RpiT1)) from Ralstonia pickettii T1 adsorbs to PHB surface via its substrate-binding domain (SBD) to enhance PHB degradation. Our previous study combining PCR random mutagenesis with the determination of PHB degradation levels of mutant enzymes suggested that Ser, Tyr, Val, Ala, and Leu residues in SBD are probably involved in the enzymatic adsorption to and degradation of PHB. In the present study, the effects of mutations at Leu441, Tyr443, and Ser445 on PHB degradation were investigated because these residues were predicted to form a beta-sheet structure and orient in the same direction to interact possibly directly with the PHB surface. Purified L441H, Y443H, and S445C mutant enzymes were prepared, and their CD spectra and hydrolytic activities for water-soluble substrates were found to be identical to those of wild-type enzyme, indicating that these mutations have no influence on their structures and their ability to cleave the ester bond. In contrast, the PHB-degrading activity of these mutants differed from that of the wild type: L441H and Y443H enzymes had lower PHB-degrading activity than their wild-type counterpart, whereas S445C had higher activity. Kinetic analysis of PHB degradation by the mutants suggested that the hydrophobic residues at these positions are important for the enzyme adsorption to the PHB surface, and such substitutions as Y443H and S445C may more effectively disrupt the PHB surface to enhance the hydrolysis of PHB polymer chains than the wild-type enzyme. Surface plasmon resonance (SPR) analysis revealed that the three substitutions mentioned above altered the association phase rather than the dissociation phase in the enzyme adsorption to the polymer surface.

  6. The Crystal Structure of the PB2 Cap-binding Domain of Influenza B Virus Reveals a Novel Cap Recognition Mechanism*

    PubMed Central

    Liu, Yong; Yang, Yongfeng; Fan, Jialin; He, Ruina; Luo, Ming; Zheng, Xiaofeng

    2015-01-01

    The influenza RNA-dependent RNA polymerase is a core enzyme required for both transcription and replication of the virus RNA genome, making it a potential drug target for the influenza virus. To detect the feature of cap-dependent transcription of influenza B virus (FluB) polymerase, we determined the crystal structures of the wild-type FluB polymerase PB2 subunit cap-binding domain (PB2cap) with bound GDP and the mutant FluB Q325F PB2cap with bound m7GDP or GDP. These structures revealed that, distinct from influenza A virus (FluA) PB2cap, the guanine and ribose moieties of substrates invert in FluB PB2caps. Moreover, we characterized the substrate specificity and affinity of the PB2caps using isothermal titration calorimetry. FluB PB2cap has a weaker affinity for m7GDP than FluA PB2cap. Unlike FluA PB2cap that has a preference for m7GDP in comparison with GDP, FluB PB2cap shows an analogous affinity for both substrates. Replacement of FluB PB2 Glu325 by Phe, the corresponding residue of FluA PB2, increased the binding affinity of FluB PB2cap for m7GDP to a level approximate to that of FluA PB2cap and caused a significant higher affinity to GDP. This study indicated that FluB PB2cap has a unique cap recognition mechanism compared with FluA PB2cap, providing molecular insight into inhibitor design targeting FluB PB2cap. PMID:25691568

  7. Piping Flexibility

    NASA Technical Reports Server (NTRS)

    1978-01-01

    A NASA computer program aids Hudson Engineering Corporation, Houston, Texas, in the design and construction of huge petrochemical processing plants like the one shown, which is located at Ju'aymah, Saudi Arabia. The pipes handling the flow of chemicals are subject to a variety of stresses, such as weight and variations in pressure and temperature. Hudson Engineering uses a COSMIC piping flexibility analysis computer program to analyze stresses and unsure the necessary strength and flexibility of the pipes. This program helps the company realize substantial savings in reduced engineering time.

  8. Flexible flatfoot

    PubMed Central

    Atik, Aziz; Ozyurek, Selahattin

    2014-01-01

    While being one of the most frequent parental complained deformities, flatfoot does not have a universally accepted description. The reasons of flexible flatfoot are still on debate, but they must be differentiated from rigid flatfoot which occurs secondary to other pathologies. These children are commonly brought up to a physician without any complaint. It should be kept in mind that the etiology may vary from general soft tissue laxities to intrinsic foot pathologies. Every flexible flatfoot does not require radiological examination or treatment if there is no complaint. Otherwise further investigation and conservative or surgical treatment may necessitate. PMID:28058304

  9. A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

    SciTech Connect

    Breustedt, Daniel A.; Chatwell, Lorenz; Skerra, Arne

    2009-10-01

    The crystal structure of tear lipocalin determined in space group P2{sub 1} revealed large structural deviations from the previously solved X-ray structure in space group C2, especially in the loop region and adjoining parts of the β-barrel which give rise to the ligand-binding site. These findings illustrate a novel mechanism for promiscuity in ligand recognition by the lipocalin protein family. Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2{sub 1} with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2{sub 1} crystal form uncovered major conformational changes (i) in β-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the β-barrel and (iii) in the extended C-terminal segment, which is attached to the β-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

  10. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3.

    PubMed

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan Dw

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

  11. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

    PubMed Central

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan DW

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed. PMID:24637704

  12. The 1.75 Å resolution structure of fission protein Fis1 from Saccharomyces cerevisiae reveals elusive interactions of the autoinhibitory domain.

    PubMed

    Tooley, James E; Khangulov, Victor; Lees, Jonathan P B; Schlessman, Jamie L; Bewley, Maria C; Heroux, Annie; Bosch, Jürgen; Hill, R Blake

    2011-11-01

    Fis1 mediates mitochondrial and peroxisomal fission. It is tail-anchored to these organelles by a transmembrane domain, exposing a soluble cytoplasmic domain. Previous studies suggested that Fis1 is autoinhibited by its N-terminal region. Here, a 1.75 Å resolution crystal structure of the Fis1 cytoplasmic domain from Saccharomyces cerevisiae is reported which adopts a tetratricopeptide-repeat fold. It is observed that this fold creates a concave surface important for fission, but is sterically occluded by its N-terminal region. Thus, this structure provides a physical basis for autoinhibition and allows a detailed examination of the interactions that stabilize the inhibited state of this molecule.

  13. Characterization of the Lipid Binding Properties of Otoferlin Reveals Specific Interactions between PI(4,5)P2 and the C2C and C2F Domains

    PubMed Central

    2015-01-01

    Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30–50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25–95 μM) and low affinities (Kd = 400–700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B–C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1′-myoinositol-4′,5′-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium

  14. Molecular Dynamics Simulations Reveal the Conformational Flexibility of Lipid II and Its Loose Association with the Defensin Plectasin in the Staphylococcus aureus Membrane.

    PubMed

    Witzke, Sarah; Petersen, Michael; Carpenter, Timothy S; Khalid, Syma

    2016-06-14

    Lipid II is critical for peptidoglycan synthesis, which is the main component of the bacterial cell wall. Lipid II is a relatively conserved and important part of the cell wall biosynthesis pathway and is targeted by antibiotics such as the lantibiotics, which achieve their function by disrupting the biosynthesis of the cell wall. Given the urgent need for development of novel antibiotics to counter the growing threat of bacterial infection resistance, it is imperative that a thorough molecular-level characterization of the molecules targeted by antibiotics be achieved. To this end, we present a molecular dynamics simulation study of the conformational dynamics of Lipid II within a detailed model of the Staphylococcus aureus cell membrane. We show that Lipid II is able to adopt a range of conformations, even within the packed lipidic environment of the membrane. Our simulations also reveal dimerization of Lipid II mediated by cations. In the presence of the defensin peptide plectasin, the conformational lability of Lipid II allows it to form loose complexes with the protein, via a number of different binding modes.

  15. Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state

    SciTech Connect

    Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T.

    2014-03-01

    This work reports the first sub-angstrom resolution structure of S. erythraeus trypsin. The detailed model of a prototypical serine protease at a catalytically relevant pH with an unoccupied active site is presented and is compared with other high-resolution serine protease structures. With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

  16. In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions

    PubMed Central

    Melnick, Ari; Ahmad, K. Farid; Arai, Sally; Polinger, Adam; Ball, Helen; Borden, Katherine L.; Carlile, Graeme W.; Prive, Gilbert G.; Licht, Jonathan D.

    2000-01-01

    The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression. PMID:10938130

  17. CW-EPR studies revealed different motional properties and oligomeric states of the integrin β1a transmembrane domain in detergent micelles or liposomes

    PubMed Central

    Yu, Lu; Wang, Wei; Ling, Shenglong; Liu, Sanling; Xiao, Liang; Xin, Yanlong; Lai, Chaohua; Xiong, Ying; Zhang, Longhua; Tian, Changlin

    2015-01-01

    Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or β subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin β1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin β1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin β1a-TM regions with different motional properties in micelles and a non-continuous integrin β1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin β1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin β1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin β1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes. PMID:25597475

  18. Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes.

    PubMed

    Walter, Justin D; Werther, Rachel A; Brison, Caileen M; Cragerud, Rebecca K; Healey, John F; Meeks, Shannon L; Lollar, Pete; Spiegel, P Clint

    2013-12-19

    The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously was investigated by x-ray crystallography. The C2 domain/3E6 FAB/G99 FAB ternary complex illustrates that each antibody recognizes epitopes on opposing faces of the factor VIII C2 domain. The 3E6 epitope forms direct contacts to the C2 domain at 2 loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, whereas the G99 epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282, and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charge present on both C2 epitopes and complementary negative charge on each antibody. A new model of membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. These results illustrate the potential complexities of the polyclonal anti-factor VIII immune response and further define the "classical" and "nonclassical" types of antibody inhibitors against the factor VIII C2 domain.

  19. Spatial and temporal variability of biomarkers and microbial diversity reveal metabolic and community flexibility in Streamer Biofilm Communities in the Lower Geyser Basin, Yellowstone National Park.

    PubMed

    Schubotz, F; Meyer-Dombard, D R; Bradley, A S; Fredricks, H F; Hinrichs, K-U; Shock, E L; Summons, R E

    2013-11-01

    Detailed analysis of 16S rRNA and intact polar lipids (IPLs) from streamer biofilm communities (SBCs), collected from geochemically similar hot springs in the Lower Geyser Basin, Yellowstone National Park, shows good agreement and affirm that IPLs can be used as reliable markers for the microbial constituents of SBCs. Uncultured Crenarchaea are prominent in SBS, and their IPLs contain both glycosidic and mixed glyco-phospho head groups with tetraether cores, having 0-4 rings. Archaeal IPL contributions increase with increasing temperature and comprise up to one-fourth of the total IPL inventory at >84 °C. At elevated temperatures, bacterial IPLs contain abundant glycosidic glycerol diether lipids. Diether and diacylglycerol (DAG) lipids with aminopentanetetrol and phosphatidylinositol head groups were identified as lipids diagnostic of Aquificales, while DAG glycolipids and glyco-phospholipids containing N-acetylgycosamine as head group were assigned to members of the Thermales. With decreasing temperature and concomitant changes in water chemistry, IPLs typical of phototrophic bacteria, such as mono-, diglycosyl, and sulfoquinovosyl DAG, which are specific for cyanobacteria, increase in abundance, consistent with genomic data from the same samples. Compound-specific stable carbon isotope analysis of IPL breakdown products reveals a large isotopic diversity among SBCs in different hot springs. At two of the hot springs, 'Bison Pool' and Flat Cone, lipids derived from Aquificales are enriched in (13) C relative to biomass and approach values close to dissolved inorganic carbon (DIC) (approximately 0‰), consistent with fractionation during autotrophic carbon fixation via the reversed tricarboxylic acid pathway. At a third site, Octopus Spring, the same Aquificales-diagnostic lipids are 10‰ depleted relative to biomass and resemble stable carbon isotope values of dissolved organic carbon (DOC), indicative of heterotrophy. Other bacterial and archaeal lipids show

  20. Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca(2+)-binding affinity in a C-domain mutant of cardiac troponin C.

    PubMed

    Veltri, Tiago; de Oliveira, Guilherme A P; Bienkiewicz, Ewa A; Palhano, Fernando L; Marques, Mayra de A; Moraes, Adolfo H; Silva, Jerson L; Sorenson, Martha M; Pinto, Jose R

    2017-04-06

    The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca(2+)-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca(2+) than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca(2+)-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

  1. The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

    PubMed

    Manso, Yasmina; Serra, Montserrat; Comes, Gemma; Giralt, Mercedes; Carrasco, Javier; Cols, Neus; Vasák, Milan; González-Duarte, Pilar; Hidalgo, Juan

    2010-06-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

  2. Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases.

    PubMed

    Handa, P; Acharya, N; Varshney, U

    2001-05-18

    Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.

  3. Local twin domains and tip-voltage-induced domain switching of monoclinic MC phase in Pb(Mg1/3Nb2/3)O3-0.34PbTiO3 single crystal revealed by piezoresponse force microscopy

    DOE PAGES

    Wang, Ruixue; Yang, Bin; Luo, Zhenlin; ...

    2016-08-29

    Here, the monoclinic (M) phases in high-performance relaxor-based ferroelectric single crystals have been recognized to be a vital structural factor for the outstanding piezoelectric property. However, due to the complexity of the structure in M phases, the understanding about it is still limited. In this paper, the local twin domains and tip-voltage-induced domain switching of the MC phase in Pb(Mg1/3Nb2/3)O3 - 0.34PbTiO3 (PMN-0.34PT) single crystal have been intensively investigated by piezoresponse force microscopy (PFM). By theoretically analyzing the experimental patterns of domain walls on the (001)C face, the specific MC twin domains in the initial annealed state of a selectedmore » area have been clarified, and the polarization orientation of the MC phase in this sample is determined to be at an angle of 29 degrees to the < 001 > C directions. In addition, based on the evolution of domains and the motion of domain walls under the step-increased PFM tip dc voltage (Vdc), the switching process and features of different types of MC domain variants are visually revealed« less

  4. Local twin domains and tip-voltage-induced domain switching of monoclinic MC phase in Pb (M g1 /3N b2 /3) O3-0.34 PbTi O3 single crystal revealed by piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Ruixue; Yang, Bin; Luo, Zhenlin; Sun, Enwei; Sun, Yuan; Xu, Han; Zhao, Jiangtao; Zheng, Limei; Zhou, Hua; Gao, Chen; Cao, Wenwu

    2016-08-01

    The monoclinic (M) phases in high-performance relaxor-based ferroelectric single crystals have been recognized to be a vital structural factor for the outstanding piezoelectric property. However, due to the complexity of the structure in M phases, the understanding about it is still limited. In this paper, the local twin domains and tip-voltage-induced domain switching of the MC phase in Pb (M g1 /3N b2 /3) O3-0.34 PbTi O3 (PMN-0.34PT) single crystal have been intensively investigated by piezoresponse force microscopy (PFM). By theoretically analyzing the experimental patterns of domain walls on the (001) C face, the specific MC twin domains in the initial annealed state of a selected area have been clarified, and the polarization orientation of the MC phase in this sample is determined to be at an angle of 29∘ to the <001> C directions. In addition, based on the evolution of domains and the motion of domain walls under the step-increased PFM tip dc voltage (Vdc), the switching process and features of different types of MC domain variants are visually revealed.

  5. The 1.75 Å resolution structure of fission protein Fis1 from Saccharomyces cerevisiae reveals elusive interactions of the autoinhibitory domain

    PubMed Central

    Tooley, James E.; Khangulov, Victor; Lees, Jonathan P. B.; Schlessman, Jamie L.; Bewley, Maria C.; Heroux, Annie; Bosch, Jürgen; Hill, R. Blake

    2011-01-01

    Fis1 mediates mitochondrial and peroxisomal fission. It is tail-anchored to these organelles by a transmembrane domain, exposing a soluble cytoplasmic domain. Previous studies suggested that Fis1 is autoinhibited by its N-terminal region. Here, a 1.75 Å resolution crystal structure of the Fis1 cytoplasmic domain from Saccharomyces cerevisiae is reported which adopts a tetratricopeptide-repeat fold. It is observed that this fold creates a concave surface important for fission, but is sterically occluded by its N-terminal region. Thus, this structure provides a physical basis for autoinhibition and allows a detailed examination of the interactions that stabilize the inhibited state of this molecule. PMID:22102223

  6. Structures of the HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of the AIM2 Inflammasome and IFI16 Receptor

    SciTech Connect

    Jin, Tengchuan; Perry, Andrew; Jiang, Jiansheng; Smith, Patrick; Curry, James A.; Unterholzner, Leonie; Jiang, Zhaozhao; Horvath, Gabor; Rathinam, Vijay A.; Johnstone, Ricky W.; Hornung, Veit; Latz, Eicke; Bowie, Andrew G.; Fitzgerald, Katherine A.; Xiao, T. Sam

    2012-05-21

    Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.

  7. Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

    PubMed Central

    De Caroli, Monica; Lenucci, Marcello S.; Manualdi, Francesca; Dalessandro, Giuseppe; De Lorenzo, Giulia; Piro, Gabriella

    2015-01-01

    The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal. PMID:26379688

  8. Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development.

    PubMed

    Macatee, Timothy L; Hammond, Benjamin P; Arenkiel, Benjamin R; Francis, Lily; Frank, Deborah U; Moon, Anne M

    2003-12-01

    Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.

  9. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

    PubMed Central

    Theodoridou, Maria; Nomikos, Michail; Parthimos, Dimitris; Gonzalez-Garcia, J. Raul; Elgmati, Khalil; Calver, Brian L.; Sideratou, Zili; Nounesis, George; Swann, Karl; Lai, F. Anthony

    2013-01-01

    Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca2+ oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca2+ in eggs. To investigate how discrete protein domains contribute to Ca2+ release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca2+ oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca2+ sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca2+ oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca2+ releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca2+ sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca2+ oscillations in eggs, we have developed a mathematical model that incorporates Ca2+-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca2+ oscillatory activity associated with specific PLC variants. PMID:24152875

  10. The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

    PubMed Central

    Krishna, S. Sri; Aravind, L.; Bakolitsa, Constantina; Caruthers, Jonathan; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L.; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0). PMID:20944206

  11. Structural and Functional Analysis of the Spt16p N-Terminal Domain Reveals Overlapping Roles of yFACT Subunits

    SciTech Connect

    VanDemark,A.; Xin, H.; McCullough, L.; Rawlins, R.; Bentley, S.; Heroux, A.; Stillman, D.; Hill, C.; Formosa, T.

    2008-01-01

    yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.

  12. Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding.

    PubMed

    Chen, Wei-Fei; Dai, Yang-Xue; Duan, Xiao-Lei; Liu, Na-Nv; Shi, Wei; Li, Na; Li, Ming; Dou, Shou-Xing; Dong, Yu-Hui; Rety, Stephane; Xi, Xu-Guang

    2016-04-07

    Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity.

  13. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development.

    PubMed

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-12-05

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.

  14. Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway

    PubMed Central

    Newman, Joseph A.; Cooper, Christopher D.O.; Aitkenhead, Hazel; Gileadi, Opher

    2015-01-01

    Summary DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain. PMID:26636256

  15. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development

    PubMed Central

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-01-01

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes. PMID:27917906

  16. A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE).

    PubMed

    Degani, Genny; Altomare, Alessandra A; Colzani, Mara; Martino, Caterina; Mazzolari, Angelica; Fritz, Guenter; Vistoli, Giulio; Popolo, Laura; Aldini, Giancarlo

    2017-04-01

    The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.

  17. The crystal structure of the mycobacterium tuberculosis Rv3019c-Rv3020c ESX complex reveals a domain-swapped heterotetramer

    SciTech Connect

    Arbing, Mark A.; Kaufmann, Markus; Phan, Tung; Chan, Sum; Cascio, Duilio; Eisenberg, David

    2010-11-15

    Mycobacterium tuberculosis encodes five gene clusters (ESX-1 to ESX-5) for Type VII protein secretion systems that are implicated in mycobacterial pathogenicity. Substrates for the secretion apparatus are encoded within the gene clusters and in additional loci that lack the components of the secretion apparatus. The best characterized substrates are the ESX complexes, 1:1 heterodimers of ESAT-6 and CFP-10, the prototypical member that has been shown to be essential for Mycobacterium tuberculosis pathogenesis. We have determined the structure of EsxRS, a homolog of EsxGH of the ESX-3 gene cluster, at 1.91 {angstrom} resolution. The EsxRS structure is composed of two four-helix bundles resulting from the 3D domain swapping of the C-terminal domain of EsxS, the CFP-10 homolog. The four-helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT-6 {center_dot} CFP-10 (EsxAB) of ESX-1, but in EsxRS a hinge loop linking the {alpha}-helical domains of EsxS undergoes a loop-to-helix transition that creates the domain swapped EsxRS tetramer. Based on the atomic structure of EsxRS and existing biochemical data on ESX complexes, we propose that higher order ESX oligomers may increase avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure may be the basis of ESX complex dissociation that would free ESAT-6 to exert a cytotoxic effect.

  18. Crystal structure of the ubiquitin-like domain-CUT repeat-like tandem of special AT-rich sequence binding protein 1 (SATB1) reveals a coordinating DNA-binding mechanism.

    PubMed

    Wang, Zheng; Yang, Xue; Guo, Shuang; Yang, Yin; Su, Xun-Cheng; Shen, Yuequan; Long, Jiafu

    2014-10-03

    SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1-248, SATB1((1-248))), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.

  19. DHPLC analysis of patients with Nevoid Basal Cell Carcinoma Syndrome reveals novel PTCH missense mutations in the sterol-sensing domain.

    PubMed

    Marsh, A; Wicking, C; Wainwright, B; Chenevix-Trench, G

    2005-09-01

    Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

  20. Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin.

    PubMed

    Osborn, Kenneth D; Bartlett, Ryan K; Mandal, Abhijit; Zaidi, Asma; Urbauer, Ramona J Bieber; Urbauer, Jeffrey L; Galeva, Nadya; Williams, Todd D; Johnson, Carey K

    2004-10-12

    We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.

  1. Cell-to-cell movement of green fluorescent protein reveals post-phloem transport in the outer integument and identifies symplastic domains in Arabidopsis seeds and embryos.

    PubMed

    Stadler, Ruth; Lauterbach, Christian; Sauer, Norbert

    2005-10-01

    Developing Arabidopsis (Arabidopsis thaliana) seeds and embryos represent a complex set of cell layers and tissues that mediate the transport and partitioning of carbohydrates, amino acids, hormones, and signaling molecules from the terminal end of the funicular phloem to and between these seed tissues and eventually to the growing embryo. This article provides a detailed analysis of the symplastic domains and the cell-to-cell connectivity from the end of the funiculus to the embryo, and within the embryo during its maturation. The cell-to-cell movement of the green fluorescent protein or of mobile and nonmobile green fluorescent protein fusions was monitored in seeds and embryos of plants expressing the corresponding cDNAs under the control of various promoters (SUC2, SUC3, TT12, and GL2) shown to be active in defined seed or embryo cell layers (SUC3, TT12, and GL2) or only outside the developing Arabidopsis seed (AtSUC2). Cell-to-cell movement was also analyzed with the low-molecular-weight fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate. The analyses presented identify a phloem-unloading domain at the end of the funicular phloem, characterize the entire outer integument as a symplastic extension of the phloem, and describe the inner integument and the globular stage embryo plus the suspensor as symplastic domains. The results also show that, at the time of hypophysis specification, the symplastic connectivity between suspensor and embryo is reduced or interrupted and that the embryo develops from a single symplast (globular and heart stage) to a mature embryo with new symplastic domains.

  2. tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features.

    PubMed Central

    Marck, Christian; Grosjean, Henri

    2002-01-01

    From 50 genomes of the three domains of life (7 eukarya, 13 archaea, and 30 bacteria), we extracted, analyzed, and compared over 4,000 sequences corresponding to cytoplasmic, nonorganellar tRNAs. For each genome, the complete set of tRNAs required to read the 61 sense codons was identified, which permitted revelation of three major anticodon-sparing strategies. Other features and sequence peculiarities analyzed are the following: (1) fit to the standard cloverleaf structure, (2) characteristic consensus sequences for elongator and initiator tDNAs, (3) frequencies of bases at each sequence position, (4) type and frequencies of conserved 2D and 3D base pairs, (5) anticodon/tDNA usages and anticodon-sparing strategies, (6) identification of the tRNA-Ile with anticodon CAU reading AUA, (7) size of variable arm, (8) occurrence and location of introns, (9) occurrence of 3'-CCA and 5'-extra G encoded at the tDNA level, and (10) distribution of the tRNA genes in genomes and their mode of transcription. Among all tRNA isoacceptors, we found that initiator tDNA-iMet is the most conserved across the three domains, yet domain-specific signatures exist. Also, according to which tRNA feature is considered (5'-extra G encoded in tDNAs-His, AUA codon read by tRNA-Ile with anticodon CAU, presence of intron, absence of "two-out-of-three" reading mode and short V-arm in tDNA-Tyr) Archaea sequester either with Bacteria or Eukarya. No common features between Eukarya and Bacteria not shared with Archaea could be unveiled. Thus, from the tRNomic point of view, Archaea appears as an "intermediate domain" between Eukarya and Bacteria. PMID:12403461

  3. Crystal structures reveal N-terminal Domain of Arabidopsis thaliana ClpD to be highly divergent from that of ClpC1

    PubMed Central

    Mohapatra, Chinmayee; Kumar Jagdev, Manas; Vasudevan, Dileep

    2017-01-01

    The caseinolytic protease machinery associated chaperone protein ClpC is known to be present in bacteria, plants and other eukaryotes, whereas ClpD is unique to plants. Plant ClpC and ClpD proteins get localized into chloroplast stroma. Herein, we report high resolution crystal structures of the N-terminal domain of Arabidopsis thaliana ClpC1 and ClpD. Surprisingly, AtClpD, but not AtClpC1, deviates from the typical N-terminal repeat domain organization of known Clp chaperones and have only seven α-helices, instead of eight. In addition, the loop connecting the two halves of AtClpD NTD is longer and covers the region which in case of AtClpC1 is thought to contribute to adaptor protein interaction. Taken together, the N-terminal domain of AtClpD has a divergent structural organization compared to any known Clp chaperones which hints towards its specific role during plant stress conditions, as opposed to that in the maintenance of chloroplastic homeostasis by AtClpC1. Conservation of residues in the NTD that are responsible for the binding of the cyclic peptide activator - Cyclomarin A, as reported for mycobacterial ClpC1 suggests that the peptide could be used as an activator to both AtClpC1 and AtClpD, which could be useful in their detailed in vitro functional characterization. PMID:28287170

  4. The Crystal Structure of Escherichia coli Group 4 Capsule Protein GfcC Reveals a Domain Organization Resembling That of Wza

    SciTech Connect

    Sathiyamoorthy, Karthik; Mills, Erez; Franzmann, Titus M.; Rosenshine, Ilan; Saper, Mark A.

    2012-03-15

    We report the 1.9 {angstrom} resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem {beta}-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a {beta}-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane {beta}-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.

  5. Disparate evolution of prion protein domains and the distinct origin of Doppel- and prion-related loci revealed by fish-to-mammal comparisons.

    PubMed

    Rivera-Milla, Eric; Oidtmann, Birgit; Panagiotidis, Cynthia H; Baier, Michael; Sklaviadis, Theodoros; Hoffmann, Rudolf; Zhou, Yi; Solis, Gonzalo P; Stuermer, Claudia A O; Málaga-Trillo, Edward

    2006-02-01

    Prions result from the misfolding and selective accumulation of the host-encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP-related loci directly downstream of each PrP locus, suggesting an independent origin of prion-related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular "bauplan" for all vertebrate PrPs. The C- and N-terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion-degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non-mammalian model like the zebrafish.

  6. Crystal structures reveal N-terminal Domain of Arabidopsis thaliana ClpD to be highly divergent from that of ClpC1.

    PubMed

    Mohapatra, Chinmayee; Kumar Jagdev, Manas; Vasudevan, Dileep

    2017-03-13

    The caseinolytic protease machinery associated chaperone protein ClpC is known to be present in bacteria, plants and other eukaryotes, whereas ClpD is unique to plants. Plant ClpC and ClpD proteins get localized into chloroplast stroma. Herein, we report high resolution crystal structures of the N-terminal domain of Arabidopsis thaliana ClpC1 and ClpD. Surprisingly, AtClpD, but not AtClpC1, deviates from the typical N-terminal repeat domain organization of known Clp chaperones and have only seven α-helices, instead of eight. In addition, the loop connecting the two halves of AtClpD NTD is longer and covers the region which in case of AtClpC1 is thought to contribute to adaptor protein interaction. Taken together, the N-terminal domain of AtClpD has a divergent structural organization compared to any known Clp chaperones which hints towards its specific role during plant stress conditions, as opposed to that in the maintenance of chloroplastic homeostasis by AtClpC1. Conservation of residues in the NTD that are responsible for the binding of the cyclic peptide activator - Cyclomarin A, as reported for mycobacterial ClpC1 suggests that the peptide could be used as an activator to both AtClpC1 and AtClpD, which could be useful in their detailed in vitro functional characterization.

  7. FLEXIBLE COUPLING

    DOEpatents

    Babelay, E.F.

    1962-02-13

    A flexible shaft coupling for operation at speeds in excess of 14,000 rpm is designed which requires no lubrication. A driving sleeve member and a driven sleeve member are placed in concentric spaced relationship. A torque force is transmitted to the driven member from the driving member through a plurality of nylon balls symmetrically disposed between the spaced sleeves. The balls extend into races and recesses within the respective sleeve members. The sleeve members have a suitable clearance therebetween and the balls have a suitable radial clearance during operation of the coupling to provide a relatively loose coupling. These clearances accommodate for both parallel and/or angular misalignments and avoid metal-tometal contact between the sleeve members during operation. Thus, no lubrication is needed, and a minimum of vibrations is transmitted between the sleeve members. (AEC)

  8. Structural Dynamics of the Cereblon Ligand Binding Domain

    PubMed Central

    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Hernandez Alvarez, Birte

    2015-01-01

    Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution. PMID:26024445

  9. Functional Isoforms of IκB Kinase α (IKKα) Lacking Leucine Zipper and Helix-Loop-Helix Domains Reveal that IKKα and IKKβ Have Different Activation Requirements

    PubMed Central

    McKenzie, Fergus R.; Connelly, Margery A.; Balzarano, Darlene; Müller, Jurgen R.; Geleziunas, Romas; Marcu, Kenneth B.

    2000-01-01

    The activity of the NF-κB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IκB subunits. Site-specific serine phosphorylation of IκBs by two IκB kinases (IKKα [also known as CHUK] and IKKβ) targets them for proteolysis. IKKα and -β have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic α-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKα: IKKα-ΔH and IKKα-ΔLH. IKKα-ΔH and IKKα-ΔLH are differentially spliced isoforms of the IKKα mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKα is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKα-ΔH and IKKα-ΔLH, like IKKα, respond to tumor necrosis factor alpha stimulation to potentiate NF-κB activation in HEK293 cells. A mutant, catalytically inactive form of IKKα blocked IKKα-, IKKα-ΔH-, and IKKα-ΔLH-mediated NF-κB activation. Akin to IKKα, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-κB-inducing kinase). In contrast to IKKα, IKKα-ΔLH failed to associate with either itself, IKKα, IKKβ, or NEMO-IKKγ-IKKAP1, while IKKα-ΔH complexed with IKKβ and IKKα but not with NEMO. Interestingly, each IKKα isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKα could not. IKKα-ΔCm, a recombinant mutant of IKKα structurally akin to IKKα-ΔLH, was equally functional in these assays, but in sharp contrast, IKKβ-ΔCm, a structurally analogous mutant of IKKβ, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKα and IKKβ need not be equivalent and can also exhibit

  10. Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi ▿ †

    PubMed Central

    Sultan, Syed Z.; Pitzer, Joshua E.; Boquoi, Tristan; Hobbs, Gerry; Miller, Michael R.; Motaleb, M. A.

    2011-01-01

    HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a Km of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi. PMID:21670168

  11. Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres.

    PubMed

    Alonso, Alicia; Mahmood, Radma; Li, Shulan; Cheung, Fanny; Yoda, Kinya; Warburton, Peter E

    2003-10-15

    Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres. A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation. Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA. Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal approximately 7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of approximately 7 Mb. DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3-13q33.1. The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of approximately 130, 215 and 275 kb within an approximately 6.5 Mb region. Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere 'hotspot' in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres. The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

  12. Analysis of the HD-GYP domain cyclic dimeric GMP phosphodiesterase reveals a role in motility and the enzootic life cycle of Borrelia burgdorferi.

    PubMed

    Sultan, Syed Z; Pitzer, Joshua E; Boquoi, Tristan; Hobbs, Gerry; Miller, Michael R; Motaleb, M A

    2011-08-01

    HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

  13. An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

    PubMed Central

    Fuller, S D; Bravo, R; Simons, K

    1985-01-01

    The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2990898

  14. Self-organized DNA/F-actin gels: entangled networks of nematic domains with tunable density

    NASA Astrophysics Data System (ADS)

    Butler, John; Zribi, Olena; Smalyukh, Ivan; Hwee Lai, Ghee; Golestanian, Ramin; Angelini, Thomas; Wong, Gerard

    2008-03-01

    We examine mixtures of DNA and F-actin as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network, all embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state via the osmotic pressure of uncondensed counterions, so that the inter-actin spacing within the domains decreases with increasing DNA concentration. These observations are consistent with arguments based on electrostatics and nematic elasticity.

  15. synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.

    PubMed

    Littleton, J T; Bai, J; Vyas, B; Desai, R; Baltus, A E; Garment, M B; Carlson, S D; Ganetzky, B; Chapman, E R

    2001-03-01

    Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

  16. Phage P4 origin-binding domain structure reveals a mechanism for regulation of DNA-binding activity by homo- and heterodimerization of winged helix proteins.

    PubMed

    Yeo, Hye-Jeong; Ziegelin, Günter; Korolev, Sergey; Calendar, Richard; Lanka, Erich; Waksman, Gabriel

    2002-02-01

    The origin-binding domain of the gpalpha protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gpalpha activity by the protein Cnr. We have determined the crystal structure of P4-OBD at 2.95 A resolution. The structure of P4-OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4-OBD subunit is the DNA-binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4-OBD dimer is suggested. P4-OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gpalpha dimer. P4-OBD dimerization is mediated by helices alpha1 and alpha3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA-binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo- or heterodimerization scaffold.

  17. Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase.

    PubMed

    Kirby, Tara L; Karim, Christine B; Thomas, David D

    2004-05-18

    We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.

  18. Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice.

    PubMed

    Heckman, Laura Dean; Chahrour, Maria H; Zoghbi, Huda Y

    2014-06-26

    Loss of function of the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) causes the progressive neurological disorder Rett syndrome (RTT). Conversely, duplication or triplication of Xq28 causes an equally wide-ranging progressive neurological disorder, MECP2 duplication syndrome, whose features overlap somewhat with RTT. To understand which MeCP2 functions cause toxicity in the duplication syndrome, we generated mouse models expressing endogenous Mecp2 along with a RTT-causing mutation in either the methyl-CpG binding domain (MBD) or the transcriptional repression domain (TRD). We determined that both the MBD and TRD must function for doubling MeCP2 to be toxic. Mutating the MBD reproduces the null phenotype and expressing the TRD mutant produces milder RTT phenotypes, yet both mutations are harmless when expressed with endogenous Mecp2. Surprisingly, mutating the TRD is more detrimental than deleting the entire C-terminus, indicating a dominant-negative effect on MeCP2 function, likely due to the disruption of a basic cluster.

  19. 'Escherichia Coli' MutS Tetramerization Domain Structure Reveals That Stable Dimers But Not Tetramers are Essential for DNA Mismatch Repair in Vivo

    SciTech Connect

    Mendillo, M.L.; Putnam, C.D.; Kolodner, R.D.; /UC, San Diego

    2007-07-10

    The E. coli mispair binding protein MutS forms dimers and tetramers in vitro, although the functional form in vivo is under debate. Here we demonstrate that the MutS tetramer is extended in solution using small angle x-ray scattering (SAXS) and the crystal structure of the C-terminal 34 amino acids of MutS containing the tetramer-forming domain fused to maltose binding protein (MBP). Wild-type C-terminal MBP fusions formed tetramers and could bind MutS and MutS-MutL-DNA complexes. In contrast, Asp835Arg and Arg840Glu mutations predicted to disrupt tetrameric interactions only allowed dimerization of MBP. A chromosomal MutS truncation mutation eliminating the dimerization/tetramerization domain eliminated mismatch repair, whereas the tetramer-disrupting MutS Asp835Arg and Arg840Glu mutations only modestly affected MutS function. These results demonstrate that dimerization but not tetramerization of the MutS C- terminus is essential for mismatch repair.

  20. Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.

    PubMed

    Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Gruber, Gerhard

    2017-03-07

    Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multi-domain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH and FAD domains, and the flexible linker between them is essential for optimal intra-domain crosstalk and for efficient electron transfer to the redox partner AhpC required for peroxidation.

  1. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  2. An unusual cytokine:Ig-domain interaction revealed in the crystal structure of leukemia inhibitory factor (LIF) in complex with the LIF receptor

    PubMed Central

    Huyton, Trevor; Zhang, Jian-Guo; Luo, Cindy S.; Lou, Mei-Zhen; Hilton, Douglas J.; Nicola, Nicos A.; Garrett, Thomas P. J.

    2007-01-01

    Leukemia inhibitory factor (LIF) receptor is a cell surface receptor that mediates the actions of LIF and other IL-6 type cytokines through the formation of high-affinity signaling complexes with gp130. Here we present the crystal structure of a complex of mouse LIF receptor with human LIF at 4.0 Å resolution. The structure is, to date, the largest cytokine receptor fragment determined by x-ray crystallography. The binding of LIF to its receptor via the central Ig-like domain is unlike other cytokine receptor complexes that bind ligand predominantly through their cytokine-binding modules. This structure, in combination with previous crystallographic studies, also provides a structural template to understand the formation and orientation of the high-affinity signaling complex between LIF, LIF receptor, and gp130. PMID:17652170

  3. The self-inhibited structure of full-length PCSK9 at 1.9 A reveals structural homology with resistin within the C-terminal domain.

    PubMed

    Hampton, Eric N; Knuth, Mark W; Li, Jun; Harris, Jennifer L; Lesley, Scott A; Spraggon, Glen

    2007-09-11

    Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-A resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.

  4. Inherent chaperone-like activity of aspartic proteases reveals a distant evolutionary relation to double-ψ barrel domains of AAA-ATPases

    PubMed Central

    Hulko, Michael; Lupas, Andrei N.; Martin, Jörg

    2007-01-01

    Chaperones and proteases share the ability to interact with unfolded proteins. Here we show that enzymatically inactive forms of the aspartic proteases HIV-1 protease and pepsin have inherent chaperone-like activity and can prevent the aggregation of denatured substrate proteins. In contrast to proteolysis, which requires dimeric enzymes, chaperone-like activity could be observed also with monomeric domains. The involvement of the active site cleft in the chaperone-like function was demonstrated by the inhibitory effect of peptide substrate inhibitors. The high structural similarity between aspartic proteases and the N-terminal double-ψ barrels of Cdc48-like proteins, which are involved in the unfolding and dissociation of proteins, suggests that they share a common ancestor. The latent chaperone-like activity in aspartic proteases can be seen as a relic that has further evolved to serve substrate binding in the context of proteolytic activity. PMID:17384229

  5. Structural and functional characterization of the 2H-phosphatase domain of Sts-2 reveals an acid-dependent phosphatase activity.

    PubMed

    Chen, Yunting; Jakoncic, Jean; Carpino, Nick; Nassar, Nicolas

    2009-03-03

    The suppressors of T cell receptor (TCR) signaling 1 and 2 (Sts-1 and -2, respectively) are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including TCR and the epidermal growth factor receptor (EGFR). Sts-1 was recently shown to be a new type of protein tyrosine phosphatase (PTP), with the phosphatase activity located within its C-terminal phosphoglycerate mutase (PGM) homology domain and key for the regulation of TCR signaling in T cells. The activity of the related Sts-2 enzyme is significantly less than that of Sts-1. Here we investigate the phosphatase activity of the PGM domain of Sts-2, Sts-2(PGM). The crystal structure of Sts-2(PGM) is remarkably similar to Sts-1(PGM), including conservation of all catalytic residues. Insight into mechanistic details is provided by the structures of the apo, tungstate-bound, and phosphate-bound enzyme. The active site shows stringent specificity, with the k(cat) optimum at pH 5.0 suggesting that Sts-2 might function as an acid-dependent phosphatase. Mutation of active site residues Gln372, Ala446, Glu481, Ser552, and Ser582 to their equivalents in Sts-1 increases the phosphatase activity of Sts-2(PGM) toward model substrates. Overall, our data demonstrate that Sts-2(PGM) adopts the conformation of an active phosphatase whose activity is fundamentally different from that of Sts-1 despite the strong structural homology. They also demonstrate that nonconserved active site residues are responsible for the difference in activity between the two isoforms. These differences reflect possible distinct physiological substrates.

  6. Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

    SciTech Connect

    Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko

    2009-12-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

  7. An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

    PubMed Central

    Fisher, K E; Eisenstein, E

    1993-01-01

    High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation. Images PMID:8407838

  8. Comparative Genomics of Two Closely Related Unicellular Thermo-Acidophilic Red Algae, Galdieria sulphuraria and Cyanidioschyzon merolae, Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae1

    PubMed Central

    Barbier, Guillaume; Oesterhelt, Christine; Larson, Matthew D.; Halgren, Robert G.; Wilkerson, Curtis; Garavito, R. Michael; Benning, Christoph; Weber, Andreas P.M.

    2005-01-01

    Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria. PMID:15710685

  9. Conformational flexibility in biochemical regulation

    SciTech Connect

    Trewhella, J.

    1993-09-01

    Small-angle X-ray and neutron scattering have proven extremely useful for studying the evolutionarily related dumbbell-shaped Ca {sup 2+} -binding proteins calmodulin and troponin C and their interactions with the target proteins whose activity they regulate. Calmodulin contracts about target enzyme binding domains with the common characteristic of having a high propensity for forming a basic, amphipathic a-helix. The contraction is achieved via flexibility in the interconnecting helix region of the molecule that links its two globular domains. This flexibility allows calmodulin to optimize its binding to different arrangements of hydrophobic and charged residues important in forming these complexes. In contrast calmodulin remains extended in its interaction with the catalytic subunit of phosphorylase kinase. There are structural and functional similarities between this interaction and that of troponin C and troponin I. Our most recent neutron scattering experiments confirm our prediction that troponin C also remains extended in this complex. The ability of the dumbbell-shaped Ca {sup 2+} -binding proteins to modulate their conformations via flexibility in the interconnecting helix region in order to accommodate different target binding domains is a remarkable example nature building functional diversity as well as specificity into a compact and unusual shape.

  10. Structural properties of PAS domains from the KCNH potassium channels.

    PubMed

    Adaixo, Ricardo; Harley, Carol A; Castro-Rodrigues, Artur F; Morais-Cabral, João H

    2013-01-01

    KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.

  11. Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

    SciTech Connect

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Ling; Robinson, Howard; Varnum, Susan M.

    2011-01-07

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR

  12. Crystal Structure of the Receptor Binding Domain of the botulinum C-D Mosiac Neurotoxin Reveals Potential Roles of Lysines 1118 and 1136 in Membrane Interactions

    SciTech Connect

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C ({approx}two-third) and BoNT/D ({approx}one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 {angstrom} resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal {beta}-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.

  13. Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

    PubMed Central

    Corrada, Dario; Soshilov, Anatoly A.; Denison, Michael S.

    2016-01-01

    The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the

  14. A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface.

    PubMed

    Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

    2013-06-01

    The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD-CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs.

  15. The Structure of a Novel Thermophilic Esterase from the Planctomycetes Species, Thermogutta terrifontis Reveals an Open Active Site Due to a Minimal ‘Cap’ Domain

    PubMed Central

    Sayer, Christopher; Szabo, Zalan; Isupov, Michail N.; Ingham, Colin; Littlechild, Jennifer A.

    2015-01-01

    A carboxyl esterase (TtEst2) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity toward small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-acetate. The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 min at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate, and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the ‘cap’ domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalized based upon the properties of their active site pockets. PMID:26635762

  16. Allelic Analyses of the Arabidopsis YUC1 Locus Reveal Residues and Domains Essential for the Functions of YUC Family of Flavin Monooxygenases

    PubMed Central

    Hou, Xianhui; Liu, Sainan; Pierri, Florencia; Dai, Xinhua; Qu, Li-Jia; Zhao, Yunde

    2011-01-01

    Flavin monooxygenases (FMOs) play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yuc1 yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yuc1 alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide (FAD) cofactor and the other binds to nicotinamide adenine dinucleotide phosphate (NADPH). We also identified the Ser139 to Phe conversion in yuc1, a mutation that is located between the two nucleotide-binding sites. By analyzing a series of yuc1 mutants, we identified key residues and motifs essential for the functions of YUC1 FMO. PMID:21205174

  17. Revealing sub-μm and μm-scale textures in H2O ice at megabar pressures by time-domain Brillouin scattering

    PubMed Central

    Nikitin, Sergey M.; Chigarev, Nikolay; Tournat, Vincent; Bulou, Alain; Gasteau, Damien; Castagnede, Bernard; Zerr, Andreas; Gusev, Vitalyi E.

    2015-01-01

    The time-domain Brillouin scattering technique, also known as picosecond ultrasonic interferometry, allows monitoring of the propagation of coherent acoustic pulses, having lengths ranging from nanometres to fractions of a micrometre, in samples with dimension of less than a micrometre to tens of micrometres. In this study, we applied this technique to depth-profiling of a polycrystalline aggregate of ice compressed in a diamond anvil cell to megabar pressures. The method allowed examination of the characteristic dimensions of ice texturing in the direction normal to the diamond anvil surfaces with sub-micrometre spatial resolution via time-resolved measurements of the propagation velocity of the acoustic pulses travelling in the compressed sample. The achieved imaging of ice in depth and in one of the lateral directions indicates the feasibility of three-dimensional imaging and quantitative characterisation of the acoustical, optical and acousto-optical properties of transparent polycrystalline aggregates in a diamond anvil cell with tens of nanometres in-depth resolution and a lateral spatial resolution controlled by pump laser pulses focusing, which could approach hundreds of nanometres. PMID:25790808

  18. Screening in ALS and FTD patients reveals 3 novel UBQLN2 mutations outside the PXX domain and a pure FTD phenotype.

    PubMed

    Synofzik, Matthis; Maetzler, Walter; Grehl, Torsten; Prudlo, Johannes; Vom Hagen, Jennifer Müller; Haack, Tobias; Rebassoo, Piret; Munz, Marita; Schöls, Ludger; Biskup, Saskia

    2012-12-01

    Mutations in UBQLN2 have recently been shown to cause dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS plus frontotemporal dementia (FTD). Information on their frequency in different populations is still rare, and a pure FTD phenotype has not yet been reported. Moreover, the mutational spectrum of known UBQLN2 mutations is still limited to its PXX repeat region. Based on a screening of 206 ALS and FTD patients, we here report 3 novel UBQLN2 mutations, accounting for 1.2% (2/161) ALS and 2.2% (1/45) FTD patients, including a patient with pure FTD. All mutations were located in highly conserved domains outside the PXX repeat region and not observed in 1450 ethnically matched control X-chromosomes. All affected patients presented with apparently sporadic disease. UBQLN2 mutations are rare in Central European ALS and FTD patients, but contribute significantly to patients with seemingly sporadic disease. UBQLN2 is able to cause any disease on the ALS-FTD continuum, including pure FTD. Because the pathogenic mechanism of UBQLN2 mutations is not limited to its PXX region, UBQLN2 screening in neurodegenerative patients should not be limited to this region.

  19. Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein.

    PubMed

    Showalter, Aaron D; Smith, Timothy P L; Bennett, Gary L; Sloop, Kyle W; Whitsett, Julie A; Rhodes, Simon J

    2002-05-29

    The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

  20. The structure of α-haemoglobin in complex with a haemoglobin-binding domain from Staphylococcus aureus reveals the elusive α-haemoglobin dimerization interface.

    PubMed

    Kumar, Kaavya Krishna; Jacques, David A; Guss, J Mitchell; Gell, David A

    2014-08-01

    Adult haemoglobin (Hb) is made up of two α and two β subunits. Mutations that reduce expression of the α- or β-globin genes lead to the conditions α- or β-thalassaemia, respectively. Whilst both conditions are characterized by anaemia of variable severity, other details of their pathophysiology are different, in part owing to the greater stability of the β chains that is conferred through β self-association. In contrast, α subunits interact weakly, and in the absence of stabilizing quaternary interactions the α chain (α) is prone to haem loss and denaturation. The molecular contacts that confer weak self-association of α have not been determined previously. Here, the first structure of an α2 homodimer is reported in complex with one domain of the Hb receptor from Staphylococcus aureus. The α2 dimer interface has a highly unusual, approximately linear, arrangement of four His side chains within hydrogen-bonding distance of each other. Some interactions present in the α1β1 dimer interface of native Hb are preserved in the α2 dimer. However, a marked asymmetry is observed in the α2 interface, suggesting that steric factors limit the number of stabilizing interactions that can form simultaneously across the interface.