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Sample records for complex ino80 binds

  1. An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex.

    Science.gov (United States)

    Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C; Fischer, Alain; Durandy, Anne

    2015-04-01

    Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Phosphorylation of human INO80 is involved in DNA damage tolerance

    International Nuclear Information System (INIS)

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

    2012-01-01

    Highlights: ► Depletion of hINO80 significantly reduced PCNA ubiquitination. ► Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. ► Western blot analyses showed phosphorylated hINO80 C-terminus. ► Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

  3. INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division

    Directory of Open Access Journals (Sweden)

    Graeme J. Gowans

    2018-01-01

    Full Text Available Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the yeast metabolic cycle (YMC. Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here, we demonstrate that the INO80 chromatin-remodeling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC1-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin to restrict proliferation to metabolically optimal states.

  4. Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

    Science.gov (United States)

    Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

    2015-01-01

    ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

  5. GluR2 ligand-binding core complexes

    DEFF Research Database (Denmark)

    Kasper, C; Lunn, M-L; Liljefors, T

    2002-01-01

    X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic...

  6. Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

    Science.gov (United States)

    Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

    2014-05-02

    DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

  7. Human RAD50 makes a functional DNA-binding complex.

    Science.gov (United States)

    Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

    2015-06-01

    The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  8. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  9. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

  10. Translation initiation mediated by nuclear cap-binding protein complex.

    Science.gov (United States)

    Ryu, Incheol; Kim, Yoon Ki

    2017-04-01

    In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

  11. Dinitrogen binding and cleavage by multinuclear iron complexes.

    Science.gov (United States)

    McWilliams, Sean F; Holland, Patrick L

    2015-07-21

    The iron-molybdenum cofactor of nitrogenase has unprecedented coordination chemistry, including a high-spin iron cluster called the iron-molybdenum cofactor (FeMoco). Thus, understanding the mechanism of nitrogenase challenges coordination chemists to understand the fundamental N2 chemistry of high-spin iron sites. This Account summarizes a series of studies in which we have synthesized a number of new compounds with multiple iron atoms, characterized them using crystallography and spectroscopy, and studied their reactions in detail. These studies show that formally iron(I) and iron(0) complexes with three- and four-coordinate metal atoms have the ability to weaken and break the triple bond of N2. These reactions occur at or below room temperature, indicating that they are kinetically facile. This in turn implies that iron sites in the FeMoco are chemically reasonable locations for N2 binding and reduction. The careful evaluation of these compounds and their reaction pathways has taught important lessons about what characteristics make iron more effective for N2 activation. Cooperation of two iron atoms can lengthen and weaken the N-N bond, while three working together enables iron atoms to completely cleave the N-N bond to nitrides. Alkali metals (typically introduced into the reaction as part of the reducing agent) are thermodynamically useful because the alkali metal cations stabilize highly reduced complexes, pull electron density into the N2 unit, and make reduced nitride products more stable. Alkali metals can also play a kinetic role, because cation-π interactions with the supporting ligands can hold iron atoms near enough to one another to facilitate the cooperation of multiple iron atoms. Many of these principles may also be relevant to the iron-catalyzed Haber-Bosch process, at which collections of iron atoms (often promoted by the addition of alkali metals) break the N-N bond of N2. The results of these studies teach more general lessons as well. They

  12. Determination of binding-dioxygen in dioxygen complexes by headspace gas chromatography.

    Science.gov (United States)

    Wang, Wei; Feng, Shun; Li, Ya-ni; Wu, Meiying; Wang, Jide

    2008-06-06

    Dioxygen complexes play important roles in organisms' bodies, so the determination of binding-dioxygen has practical significance. A simple and robust method based on headspace gas chromatography was proposed to determine the binding-dioxygen in dioxygen complexes. By measuring the content change of nitrogen gas in a vial, the amount of oxygen released from dixoygen complexes can be determined. The method was validated using potassium chlorate as model sample, and the results exhibited good recoveries (90-99%) with the relative standard deviation less than 8%. It was also used to analyze dioxygen complex of cobalt bis(salicylaldehyde) ethylenediimine and polyamine cobalt complexes prepared by solid-phase reaction.

  13. Lewis base binding affinities and redox properties of plutonium complexes

    International Nuclear Information System (INIS)

    Oldham, Susan M.; Schake, Ann R.; Burns, Carol J.; Morgan, Arthur N. III; Schnabel, Richard C.; Warner, Benjamin P.; Costa, David A.; Smith, Wayne H.

    2000-01-01

    As part of the actinide molecular science competency development effort, the initial goal of this work is to synthesize and investigate several series of complexes, varying by actinide metal, ligand set, and oxidation state. We are examining the reactivity of plutonium and neptunium organometallic complexes to elucidate fundamental chemical parameters of the metals. These reactions will be compared to those of the known corresponding uranium complexes in order to recognize trends among the actinide elements and to document differences in chemical behavior

  14. Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

    Science.gov (United States)

    Fehr, Marcus; Wolf, Antje; Stammler, Gerd

    2016-03-01

    Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates. © 2015 Society of Chemical Industry.

  15. Using remote substituents to control solution structure and anion binding in lanthanide complexes

    DEFF Research Database (Denmark)

    Tropiano, Manuel; Blackburn, Octavia A.; Tilney, James A.

    2013-01-01

    A study of the anion-binding properties of three structurally related lanthanide complexes, which all contain chemically identical anion-binding motifs, has revealed dramatic differences in their anion affinity. These arise as a consequence of changes in the substitution pattern on the periphery ...

  16. Complexities in human herpesvirus-6A and -6B binding to host cells

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Höllsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...

  17. Binding of properdin to solid-phase immune complexes

    DEFF Research Database (Denmark)

    Junker, A; Baatrup, G; Svehag, S E

    1998-01-01

    The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme-linked immunosorbent assay using solid-phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake...... fixed to IC was the principal ligand for properdin in the assay. The findings could have biological implications relating to complement-mediated modification of immune complexes in disease....

  18. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    International Nuclear Information System (INIS)

    Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

    1983-01-01

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125 I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125 I-H; when fresh serum was chelated with 10 mM EDTA, 125 I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125 I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125 I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes

  19. Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

    Science.gov (United States)

    Couñago, Rafael M; Knapp, Karen M; Nakatani, Yoshio; Fleming, Stephen B; Corbett, Michael; Wise, Lyn M; Mercer, Andrew A; Krause, Kurt L

    2015-07-07

    The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a β-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP β-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP β-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

  1. Incremental binding free energies of aluminum (III) vs. magnesium (II) complexes

    International Nuclear Information System (INIS)

    Mercero, Jose M.; Mujika, Jon I.; Matxain, Jon M.; Lopez, Xabier; Ugalde, Jesus M.

    2003-01-01

    A sequential ligand addition to the aluminum (III) cation has been studied using the B3LYP functional and a combined all-electron/pseudopotentials basis set. The aluminum complexes are compared with analogous magnesium (II) complexes. Different thermodynamical data, such as incremental binding energies, enthalpies, entropies and free energies, are presented for these addition reactions. While the magnesium (II) cation can only accommodate three negatively charged ligands, aluminum (III) accommodates four even after including bulk solvent effects. The main differences between both cations complexing with the neutral ligands, is that aluminum (III) is not able to form complexes with methanol until the number of methanol ligands is equal to 3. Magnesium (II) prefers to bind methanol and formamide when the number of ligands is small, while aluminum prefers formamide. For the largest complexes both cations prefer to bind water

  2. Role of Ca2+ in the binding mechanism of EHDP-Tc complex to bone

    International Nuclear Information System (INIS)

    Langevelde, A. van; Huisman, C.M.; Driessen, O.M.J.; Pauwels, E.K.J.

    1979-01-01

    Experiments are described testing the hypothesis that 99m-Tc-EHDP complex travels to bone as a unit and dissociates at the bone binding site because of the high affinity of EHDP for hydroxyapatite after which technetium binds separately. The results indicate that technetium is not dissociated from EHDP in binding to hydroxyapatite, but the EHDP-Tc-ligand stays intact. It is postulated that calcium plays an important role in bone-labelling with EHDP-Tc complex and that in fact the EHDP-Ca-Tc complex is the binding agent. Only by assuming the presence of this agent could the action of magnesium-ions or of excess calcium-ions be explained. (Auth./C.F.)

  3. Synthesis, characterization, anti-microbial, DNA binding and cleavage studies of Schiff base metal complexes

    Directory of Open Access Journals (Sweden)

    Poomalai Jayaseelan

    2016-09-01

    Full Text Available A novel Schiff base ligand has been prepared by the condensation between butanedione monoxime with 3,3′-diaminobenzidine. The ligand and metal complexes have been characterized by elemental analysis, UV, IR, 1H NMR, conductivity measurements, EPR and magnetic studies. The molar conductance studies of Cu(II, Ni(II, Co(II and Mn(II complexes showed non-electrolyte in nature. The ligand acts as dibasic with two N4-tetradentate sites and can coordinate with two metal ions to form binuclear complexes. The spectroscopic data of metal complexes indicated that the metal ions are complexed with azomethine nitrogen and oxyimino nitrogen atoms. The binuclear metal complexes exhibit octahedral arrangements. DNA binding properties of copper(II metal complex have been investigated by electronic absorption spectroscopy. Results suggest that the copper(II complex bind to DNA via an intercalation binding mode. The nucleolytic cleavage activities of the ligand and their complexes were assayed on CT-DNA using gel electrophoresis in the presence and absence of H2O2. The ligand showed increased nuclease activity when administered as copper complex and copper(II complex behave as efficient chemical nucleases with hydrogen peroxide activation. The anti-microbial activities and thermal studies have also been studied. In anti-microbial activity all complexes showed good anti-microbial activity higher than ligand against gram positive, gram negative bacteria and fungi.

  4. Understanding TR binding to pMHC complexes: how does a TR scan many pMHC complexes yet preferentially bind to one.

    Directory of Open Access Journals (Sweden)

    Javed Mohammed Khan

    Full Text Available Understanding the basis of the binding of a T cell receptor (TR to the peptide-MHC (pMHC complex is essential due to the vital role it plays in adaptive immune response. We describe the use of computed binding (free energy (BE, TR paratope, pMHC epitope, molecular surface electrostatic potential (MSEP and calculated TR docking angle (θ to analyse 61 TR/pMHC crystallographic structures to comprehend TR/pMHC interaction. In doing so, we have successfully demonstrated a novel/rational approach for θ calculation, obtained a linear correlation between BE and θ without any "codon" or amino acid preference, provided an explanation for TR ability to scan many pMHC ligands yet specifically bind one, proposed a mechanism for pMHC recognition by TR leading to T cell activation and illustrated the importance of the peptide in determining TR specificity, challenging the "germline bias" theory.

  5. Cu(II) complexes of glyco-imino-aromatic conjugates in DNA binding ...

    Indian Academy of Sciences (India)

    Abstract. Binding of metal complexes of C2-glucosyl conjugates with DNA has been established by absorp- ... Metal complexes have shown toxicity to the HeLa and MCF–7 .... ber with 5% CO2. ..... ing/reducing agent or laser/UV–visible light.

  6. Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome bc1 Complex

    DEFF Research Database (Denmark)

    Barragan, Angela M.; Crofts, Antony R.; Schulten, Klaus

    2015-01-01

    for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol...... all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton...

  7. Variable Denticity in Carboxylate Binding to the Uranyl Coordination Complexes

    International Nuclear Information System (INIS)

    Groenewold, G.S.; De Jong, Wibe A.; Oomens, Jos; Van Stipdonk, Michael J.

    2010-01-01

    Tris-carboxylate complexes of the uranyl (UO2)2+ cation with acetate and benzoate were generated using electrospray ionization mass spectrometry, and then isolated in a Fourier transformion cyclotron resonance mass spectrometer. Wavelength-selective infrared multiple photon dissociation (IRMPD) of the tris-acetatouranyl anion resulted in a redox elimination of an acetate radical, which was used to generate an IR spectrum that consisted of six prominent absorption bands. These were interpreted with the aid of density functional theory calculations in terms of symmetric and antisymmetric -CO2 stretches of both the monodentate and bidentate acetate, CH3 bending and umbrella vibrations, and a uranyl O-U-O asymmetric stretch. The comparison of the calculated and measured IR spectra indicated that the tris-acetate complex contained two acetate ligands bound in a bidentate fashion, while the third acetate was monodentate. In similar fashion, the tris-benzoate uranyl anion was formed and photodissociated by loss of a benzoate radical, enabling measurement of the infrared spectrum that was in close agreement with that calculated for a structure containing one monodentate, and two bidentate benzoate ligands.

  8. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    Science.gov (United States)

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  10. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

  11. Synthesis, characterization, DNA binding and cleavage studies of mixed-ligand copper (II complexes

    Directory of Open Access Journals (Sweden)

    M. Sunita

    2017-05-01

    Full Text Available New two copper complexes of type [Cu(Bzimpy(LH2O]SO4 (where L = 2,2′ bipyridine (bpy, and ethylene diamine (en, Bzimpy = 2,6-bis(benzimidazole-2ylpyridine have been synthesized and characterized by elemental analyses, molar conductance measurements, magnetic susceptibility measurements, mass, IR, electronic and EPR spectral studies. Based on elemental and spectral studies six coordinated geometries were assigned to the two complexes. DNA-binding properties of these metal complexes were investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurements and thermal denaturation methods. Experimental studies suggest that the complexes bind to DNA through intercalation. These complexes also promote the cleavage of plasmid pBR322, in the presence of H2O2.

  12. Synthesis, structure, DNA/BSA binding and antibacterial studies of NNO tridentate Schiff base metal complexes

    Science.gov (United States)

    Sakthi, Marimuthu; Ramu, Andy

    2017-12-01

    A new salicylaldehyde derived 2,4-diiodo-6-((2-phenylaminoethylimino)methyl)phenol Schiff base(L) and its transition metal complexes of the type MLCl where, M = Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) have been synthesized. The coordination mode of Schiff base holding NNO donor atoms with metal ions was well investigated by elemental analysis, ESI-mass as well as IR, UV-vis, CV and NMR spectral studies. The binding efficiency and mode of these complexes with biological macromolecules viz., herring sperm DNA (HS- DNA) and bovine serum albumin (BSA) have been explored through various spectroscopic techniques. The characteristic changes in absorption, emission and, circular dichroism spectra of the complexes with DNA indicate the noticeable interaction between them. From the all spectral information complexes could interact with DNA via non-intercalation mode of binding. The hyperchromisim in absorption band and hypochromisim in emission intensity of BSA with different complex concentrations shown significant information, and the binding affinity value has been predicted from Stern-Volmer plots. Further, all the complexes could cleave the circular plasmid pUC19 DNA efficiently by using an activator H2O2. The ligand and all metal(II) complexes showed good antibacterial activities. The molecular docking studies of the complexes with DNA were performed in order to make a comparison and conclusion with spectral technic results.

  13. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  14. Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

    Science.gov (United States)

    Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

    2015-10-31

    Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

  15. Analysis of binding ability of two tetramethylpyridylporphyrins to albumin and its complex with bilirubin

    Science.gov (United States)

    Solomonov, Alexey V.; Shipitsyna, Maria K.; Vashurin, Arthur S.; Rumyantsev, Evgeniy V.; Timin, Alexander S.; Ivanov, Sergey P.

    2016-11-01

    An interaction between 5,10,15,20-tetrakis-(N-methyl-x-pyridyl)porphyrins, x = 2; 4 (TMPyPs) with bovine serum albumin (BSA) and its bilirubin (BR) complex was investigated by UV-Viz and fluorescence spectroscopy under imitated physiological conditions involving molecular docking studies. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, quenching sphere radius etc.) were determined. It was showed that the interaction between proteins and TMPyPs occurs via static quenching of protein fluorescence and has predominantly hydrophobic and electrostatic character. It was revealed that obtained complexes are relatively stable, but in the case of TMPyP4 binding with proteins occurs better than TMPyP2. Nevertheless, both TMPyPs have better binding ability with free protein compared to BRBSA at the same time. The influence of TMPyPs on the conformational changes in protein molecules was studied using synchronous fluorescence spectroscopy. It was found that there is no competition of BR with TMPyPs for binging sites on protein molecule and BR displacement does not occur. Molecular docking calculations have showed that TMPyPs can bind with albumin via tryptophan residue in the hydrophilic binding site of protein molecule but it is not one possible interaction way.

  16. Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration.

    Science.gov (United States)

    Kahle, Claudia; Holzgrabe, Ulrike

    2004-10-01

    Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

  17. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

    2011-01-01

    a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

  18. Serum and plasma fibronectin binds to complement reacted immune complexes primarily via Clq

    DEFF Research Database (Denmark)

    Baatrup, G; Svehag, S E

    1986-01-01

    The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase...... with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after...

  19. Kinetically inert lanthanide complexes as reporter groups for binding of potassium by 18-crown-6

    DEFF Research Database (Denmark)

    Junker, Anne Kathrine Ravnsborg; Tropiano, Manuel; Faulkner, Stephen

    2016-01-01

    in a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction with azide-functionalized crown ethers. The resulting complexes were investigated using NMR and optical methods. Titrations with potassium chloride in methanol observing the sensititzed europium- and terbium-centered emissions were......-centered emission to report on the binding of potassium in an 18-crown-6 binding pocket. The responsive systems were made by linking a crown ether to a kinetically inert lanthanide binding pocket using a molecular building block approach. Specifically, an alkyne-appended Ln.DO3A was used as a building block...... used to investigate the response of the systems. The molecular reporters based on aliphatic crown ethers were found to have strongly inhibited binding of potassium, while the benzo-18-crown-6 derived systems had essentially the same association constants as the native crown ethers. The shape...

  20. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  1. Carotenoid-binding sites of the major light-harvesting complex II of higher plants

    NARCIS (Netherlands)

    Croce, Roberta; Weiss, Saskia; Bassi, Roberto

    1999-01-01

    Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron

  2. Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

    International Nuclear Information System (INIS)

    Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir; Singh, Tej P.; Mikhailov, Albert; Gabdoulkhakov, Azat; Voelter, Wolfgang; Betzel, Christian

    2004-01-01

    The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R free = 23.6%) and 20.9 (R free = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound

  3. Complexities in human herpesvirus-6A and -6B binding to host cells

    International Nuclear Information System (INIS)

    Pedersen, Simon Metz; Hoellsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher affinity than others for the virus. Within recent years, elucidation of the viral complex has identified additional HHV-6A and -6B specific glycoproteins. Thus, gH-gL associates with a gQ1-gQ2 dimer to form a heterotetrameric complex. In addition, a novel complex consisting of gH-gL-gO has been described that does not bind CD46. Accumulating evidence suggests that an additional HHV-6A and -6B receptor exists. The previous simple picture of HHV-6A/B-host cell contact therefore includes more layers of complexities on both the viral and the host cell side of the interaction

  4. A Mononuclear Non-Heme Manganese(IV)-Oxo Complex Binding Redox-Inactive Metal Ions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Junying; Lee, Yong-Min; Davis, Katherine M.; Wu, Xiujuan; Seo, Mi Sook; Cho, Kyung-Bin; Yoon, Heejung; Park, Young Jun; Fukuzumi, Shunichi; Pushkar, Yulia N.; Nam, Wonwoo [Ewha; (Purdue); (Osaka)

    2013-05-29

    Redox-inactive metal ions play pivotal roles in regulating the reactivities of high-valent metal–oxo species in a variety of enzymatic and chemical reactions. A mononuclear non-heme Mn(IV)–oxo complex bearing a pentadentate N5 ligand has been synthesized and used in the synthesis of a Mn(IV)–oxo complex binding scandium ions. The Mn(IV)–oxo complexes were characterized with various spectroscopic methods. The reactivities of the Mn(IV)–oxo complex are markedly influenced by binding of Sc3+ ions in oxidation reactions, such as a ~2200-fold increase in the rate of oxidation of thioanisole (i.e., oxygen atom transfer) but a ~180-fold decrease in the rate of C–H bond activation of 1,4-cyclohexadiene (i.e., hydrogen atom transfer). The present results provide the first example of a non-heme Mn(IV)–oxo complex binding redox-inactive metal ions that shows a contrasting effect of the redox-inactive metal ions on the reactivities of metal–oxo species in the oxygen atom transfer and hydrogen atom transfer reactions.

  5. Synthesis and structure elucidation of a copper(II) Schiff-base complex: in vitro DNA binding, pBR322 plasmid cleavage and HSA binding studies.

    Science.gov (United States)

    Tabassum, Sartaj; Ahmad, Musheer; Afzal, Mohd; Zaki, Mehvash; Bharadwaj, Parimal K

    2014-11-01

    New copper(II) complex with Schiff base ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid (H₂L) was synthesized and characterized by spectroscopic and analytical and single crystal X-ray diffraction studies which revealed that the complex 1 exist in a distorted octahedral environment. In vitro CT-DNA binding studies were performed by employing different biophysical technique which indicated that the 1 strongly binds to DNA in comparison to ligand via electrostatic binding mode. Complex 1 cleaves pBR322 DNA via hydrolytic pathway and recognizes minor groove of DNA double helix. The HSA binding results showed that ligand and complex 1 has ability to quench the fluorescence emission intensity of Trp 214 residue available in the subdomain IIA of HSA. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Colloidal gold-labeled insulin complex. Characterization and binding to adipocytes.

    Science.gov (United States)

    Moll, U M; Thun, C; Pfeiffer, E F

    1986-01-01

    Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10-15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.

  7. Comparison and analysis on the serum-binding characteristics of aspirin-zinc complex and aspirin.

    Science.gov (United States)

    Zhang, Hua-Xin; Zhang, Qun; Wang, Hong-Lin; Li, Li-Wei

    2017-09-01

    This study was designed to compare the protein-binding characteristics of aspirin-zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three-dimensional fluorescence, ultraviolet-visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non-radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA-binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α-helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

    Science.gov (United States)

    Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

    2016-10-10

    The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

  9. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: ehmke.pohl@durham.ac.uk [Durham University, (United Kingdom); Usón, Isabel, E-mail: ehmke.pohl@durham.ac.uk [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)

    2014-06-01

    The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  10. Cu(II) Complexes of Isoniazid Schiff Bases: DNA/BSA Binding and Cytotoxicity Studies on A549 Cell Line

    OpenAIRE

    Ramadevi, Pulipaka; Singh, Rinky; Prajapati, Akhilesh; Gupta, Sarita; Chakraborty, Debjani

    2014-01-01

    A series of isonicotinoyl hydrazones have been synthesized via template method and were complexed to Cu(II). The ligands are coordinated to Cu(II) ion through the enolic oxygen and azomethine nitrogen resulting in a square planar geometry. The CT-DNA and bovine serum albumin binding propensities of the compounds were determined spectrophotometrically, the results of which indicate good binding propensity of complexes to DNA and BSA with high binding constant values. Furthermore, the compounds...

  11. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    Science.gov (United States)

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

    International Nuclear Information System (INIS)

    Jacob, E.; O'Brien, H.A.W.; Mollin, D.L.

    1977-01-01

    A new radioisotope dilution assay for vitamin B 12 -intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B 12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B 12 -intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B 12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

  13. Experimental and theoretical investigation of topological and energetic characteristics of Sb complexes reversibly binding molecular oxygen.

    Science.gov (United States)

    Fukin, Georgy K; Baranov, Evgenii V; Jelsch, Christian; Guillot, Benoît; Poddel'sky, Andrey I; Cherkasov, Vladimir K; Abakumov, Gleb A

    2011-07-28

    The experimental distribution of electron density in Ph(3)(4,5-OMe-3,6-Bu(t)-Cat)Sb·MeCN (1*) and Ph(3)(4,5-N(2)C(4)H(6)-3,6-Bu(t)-Cat)Sb·MeOH (2*) complexes was studied. According to atoms in molecules theory, the Sb-C(Ph), Sb-O(catecholate), and Sb···N(O) bonds are intermediate, whereas the O-C and C-C bonds are covalent, respectively. The energy of the Sb···N(MeCN) and Sb···O(MeOH) bonds are 7.0 and 11.3 kcal/mol according to the Espinosa equation. Density functional theory and Hartree-Fock calculations were carried out for a series of catecholate and amidophenolate complexes of antimony(V). It was shown that such calculations reliably reproduce geometrical and topological parameters and therefore can be used for a criterion search of dioxygen reversible binding by the catecholate and amidophenolate complexes of antimony(V). It was found that the "critical" value of the HOMO energy vary in the range from -5.197 to -5.061 eV for reversible binding of dioxygen complexes. This can serve as a thermodynamic criterion to predict the possibility of the dioxygen reversible binding by the catecholate and amidophenolate complexes of Sb(V). The HOMO energies correlate with the conversion of the catecholate and amidophenolate complexes in corresponding spiroendoperoxide derivatives as well. The contribution of the atom orbitals of the carbon atoms in the five-membered metallocycle to HOMO in complexes with different substitutes in the 4- and 5-positions of the catecholate ligand allows predicting the place of dioxygen addition. © 2011 American Chemical Society

  14. 2,3-DPG-Hb complex: a hypothesis for an asymmetric binding.

    Science.gov (United States)

    Pomponi, M; Bertonati, C; Fuglei, E; Wiig, O; Derocher, A E

    2000-05-15

    This study was undertaken to test the symmetry of 2,3-diphosphoglycerate (2,3-DPG) binding site in hemoglobin (Hb). From Arnone's study [A. Arnone, Nature (London) 237 (1972) 146] the 2,3-DPG binding site is located at the top of the cavity, that runs through the center of the deoxy-Hb molecule. However, it is possible that this symmetry reported by Arnone, for crystals of 2,3-DPG-Hb complex, might not be conserved in solution. In this paper, we report the 31P nuclear magnetic resonances of the 2,3-DPG interaction with Hb. The 2,3-DPG chemical shifts of the P2 and P3 resonance are both pH- and hemoglobin-dependent [protein from man, polar bear (Ursus maritimus), Arctic fox (Alopex lagopus) and bovine]. 2,3-DPG binds tightly to deoxyhemoglobin and weakly, nevertheless significantly, to oxyhemoglobin. In particular, our results suggest similar spatial position of the binding site of 2,3-DPG in both forms of Hb in solutions. However, the most unexpected result was the apparent loss of symmetry in the binding site, which might correlate with the ability of the hemoglobin to modulate its functional behavior. The different interactions of the phosphate groups indicate small differences in the quaternary structure of the different deoxy forms of hemoglobin. Given the above structural perturbation an asymmetric binding in the complex could justify, at least in part, different physiological properties of Hb. Regardless, functionally relevant effects of 2,3-DPG seem to be measured and best elucidated through solution studies.

  15. Macrocyclic Gd(3+) complexes with pendant crown ethers designed for binding zwitterionic neurotransmitters.

    Science.gov (United States)

    Oukhatar, Fatima; Meudal, Hervé; Landon, Céline; Logothetis, Nikos K; Platas-Iglesias, Carlos; Angelovski, Goran; Tóth, Éva

    2015-07-27

    A series of Gd(3+) complexes exhibiting a relaxometric response to zwitterionic amino acid neurotransmitters was synthesized. The design concept involves ditopic interactions 1) between a positively charged and coordinatively unsaturated Gd(3+) chelate and the carboxylate group of the neurotransmitters and 2) between an azacrown ether appended to the chelate and the amino group of the neurotransmitters. The chelates differ in the nature and length of the linker connecting the cyclen-type macrocycle that binds the Ln(3+) ion and the crown ether. The complexes are monohydrated, but they exhibit high proton relaxivities (up to 7.7 mM(-1)  s(-1) at 60 MHz, 310 K) due to slow molecular tumbling. The formation of ternary complexes with neurotransmitters was monitored by (1) H relaxometric titrations of the Gd(3+) complexes and by luminescence measurements on the Eu(3+) and Tb(3+) analogues at pH 7.4. The remarkable relaxivity decrease (≈80 %) observed on neurotransmitter binding is related to the decrease in the hydration number, as evidenced by luminescence lifetime measurements on the Eu(3+) complexes. These complexes show affinity for amino acid neurotransmitters in the millimolar range, which can be suited to imaging concentrations of synaptically released neurotransmitters. They display good selectivity over non-amino acid neurotransmitters (acetylcholine, serotonin, and noradrenaline) and hydrogenphosphate, but selectivity over hydrogencarbonate was not achieved. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Deciphering the binding behavior of flavonoids to the cyclin dependent kinase 6/cyclin D complex.

    Directory of Open Access Journals (Sweden)

    Jingxiao Zhang

    Full Text Available Flavonoids, a class of natural compounds with variable phenolic structures, have been found to possess anti-cancer activities by modulating different enzymes and receptors like CDK6. To understand the binding behavior of flavonoids that inhibit the active CDK6, molecular dynamics (MD simulations were performed on six inhibitors, chrysin (M01, fisetin (M03, galangin (M04, genistein (M05, quercetin (M06 and kaempferol (M07, complexed with CDK6/cyclin D. For all six flavonoids, the 3'-OH and 4'-OH of B-ring were found to be favorable for hydrogen bond formation, but the 3-OH on the C-ring and 5-OH on the A-ring were unfavorable, which were confirmed by the MD simulation results of the test molecule, 3', 4', 7-trihydroxyflavone (M15. The binding efficiencies of flavonoids against the CDK6/cyclin D complex were mainly through the electrostatic (especially the H-bond force and vdW interactions with residues ILE19, VAL27, ALA41, GLU61, PHE98, GLN103, ASP163 and LEU152. The order of binding affinities of these flavonoids toward the CDK6/cyclin D was M03 > M01 > M07 > M15 > M06 > M05 > M04. It is anticipated that the binding features of flavonoid inhibitors studied in the present work may provide valuable insights for the development of CDK6 inhibitors.

  17. Computational identification of binding energy hot spots in protein-RNA complexes using an ensemble approach.

    Science.gov (United States)

    Pan, Yuliang; Wang, Zixiang; Zhan, Weihua; Deng, Lei

    2018-05-01

    Identifying RNA-binding residues, especially energetically favored hot spots, can provide valuable clues for understanding the mechanisms and functional importance of protein-RNA interactions. Yet, limited availability of experimentally recognized energy hot spots in protein-RNA crystal structures leads to the difficulties in developing empirical identification approaches. Computational prediction of RNA-binding hot spot residues is still in its infant stage. Here, we describe a computational method, PrabHot (Prediction of protein-RNA binding hot spots), that can effectively detect hot spot residues on protein-RNA binding interfaces using an ensemble of conceptually different machine learning classifiers. Residue interaction network features and new solvent exposure characteristics are combined together and selected for classification with the Boruta algorithm. In particular, two new reference datasets (benchmark and independent) have been generated containing 107 hot spots from 47 known protein-RNA complex structures. In 10-fold cross-validation on the training dataset, PrabHot achieves promising performances with an AUC score of 0.86 and a sensitivity of 0.78, which are significantly better than that of the pioneer RNA-binding hot spot prediction method HotSPRing. We also demonstrate the capability of our proposed method on the independent test dataset and gain a competitive advantage as a result. The PrabHot webserver is freely available at http://denglab.org/PrabHot/. leideng@csu.edu.cn. Supplementary data are available at Bioinformatics online.

  18. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  19. C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

    Science.gov (United States)

    Kushwaha, Ambuj K; Grove, Anne

    2013-01-24

    Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

  20. Binding mode and free energy prediction of fisetin/β-cyclodextrin inclusion complexes

    Directory of Open Access Journals (Sweden)

    Bodee Nutho

    2014-11-01

    Full Text Available In the present study, our aim is to investigate the preferential binding mode and encapsulation of the flavonoid fisetin in the nano-pore of β-cyclodextrin (β-CD at the molecular level using various theoretical approaches: molecular docking, molecular dynamics (MD simulations and binding free energy calculations. The molecular docking suggested four possible fisetin orientations in the cavity through its chromone or phenyl ring with two different geometries of fisetin due to the rotatable bond between the two rings. From the multiple MD results, the phenyl ring of fisetin favours its inclusion into the β-CD cavity, whilst less binding or even unbinding preference was observed in the complexes where the larger chromone ring is located in the cavity. All MM- and QM-PBSA/GBSA free energy predictions supported the more stable fisetin/β-CD complex of the bound phenyl ring. Van der Waals interaction is the key force in forming the complexes. In addition, the quantum mechanics calculations with M06-2X/6-31G(d,p clearly showed that both solvation effect and BSSE correction cannot be neglected for the energy determination of the chosen system.

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

    Science.gov (United States)

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

    2016-11-01

    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.

  2. Theoretical study of the binding nature of glassy carbon with nickel(II) phthalocyanine complexes

    International Nuclear Information System (INIS)

    Cortez, Luis; Berrios, Cristhian; Yanez, Mauricio; Cardenas-Jiron, Gloria I.

    2009-01-01

    A theoretical study at the semiempirical RHF/PM3(tm) level (tm: transition metal) of the binding nature between a glassy carbon (GC) cluster and a nickel(II) complex (nickel(II) phthalocyanine NiPc, nickel(II) tetrasulphophthalocyanine NiTSPc) was performed. Three types of interactions for GC...NiPc (NiTSPc) were studied: (a) through an oxo (O) bridge, (b) through an hydroxo (OH) bridge, and (c) non-bridge. One layer (NiPc, NiTSPc) and two layers (NiPc...NiPc) of complex were considered. The binding energy calculated showed that in both cases NiPc and NiTSPc, the oxo structures are more stable than the hydroxo ones, and than the non-bridge systems. Charge analysis (NAO) predicted that GC gained more electrons in an oxo structure than in the analogues hydroxo. The theoretical results showed an agreement with the experimental data available, an oxo binding between GC and a nickel complex (NiPc, NiTSPc) in aqueous alkaline solutions is formed.

  3. Theoretical study of the binding nature of glassy carbon with nickel(II) phthalocyanine complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cortez, Luis [Laboratorio de Quimica Teorica, Facultad de Quimica y Biologia, Universidad de Santiago de Chile (USACH), Casilla 40, Correo 33, Santiago (Chile); Berrios, Cristhian [Laboratorio de Electrocatalisis, Facultad de Quimica y Biologia, Universidad de Santiago de Chile (USACH), Casilla 40, Correo 33, Santiago (Chile); Yanez, Mauricio [Laboratorio de Recursos Renovables, Centro de Biotecnologia, Universidad de Concepcion, Casilla-160 C, Concepcion (Chile); Cardenas-Jiron, Gloria I., E-mail: gloria.cardenas@usach.cl [Laboratorio de Quimica Teorica, Facultad de Quimica y Biologia, Universidad de Santiago de Chile (USACH), Casilla 40, Correo 33, Santiago (Chile)

    2009-11-26

    A theoretical study at the semiempirical RHF/PM3(tm) level (tm: transition metal) of the binding nature between a glassy carbon (GC) cluster and a nickel(II) complex (nickel(II) phthalocyanine NiPc, nickel(II) tetrasulphophthalocyanine NiTSPc) was performed. Three types of interactions for GC...NiPc (NiTSPc) were studied: (a) through an oxo (O) bridge, (b) through an hydroxo (OH) bridge, and (c) non-bridge. One layer (NiPc, NiTSPc) and two layers (NiPc...NiPc) of complex were considered. The binding energy calculated showed that in both cases NiPc and NiTSPc, the oxo structures are more stable than the hydroxo ones, and than the non-bridge systems. Charge analysis (NAO) predicted that GC gained more electrons in an oxo structure than in the analogues hydroxo. The theoretical results showed an agreement with the experimental data available, an oxo binding between GC and a nickel complex (NiPc, NiTSPc) in aqueous alkaline solutions is formed.

  4. The human cap-binding complex is functionally connected to the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken Søndergaard

    2013-01-01

    Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis...

  5. SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signaling Complexes

    Directory of Open Access Journals (Sweden)

    Sebastian Kupka

    2016-08-01

    Full Text Available Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC. Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF, resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes.

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

    Science.gov (United States)

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

    2017-11-04

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

  7. Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

    Science.gov (United States)

    Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

    1998-01-01

    Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

  8. Structural analysis and binding domain of albumin complexes with natural dietary supplement humic acid

    International Nuclear Information System (INIS)

    Ding Fei; Diao Jianxiong; Yang Xinling; Sun Ying

    2011-01-01

    Humic acid, a natural ionic molecule, is rapidly being recognized as one of the crucial elements in our modern diets of the new century. A biophysical protocol utilizing circular dichroism (CD), steady state and time-resolved fluorescence for the investigation of the complexation of the humic acid to the staple in vivo transporter, human serum albumin (HSA), as a model for protein-humic substances, is proclaimed. The alterations of CD and three-dimensional fluorescence suggest that the polypeptide chain of HSA partially folded after complexation with humic acid. The data of fluorescence emission displayed that the binding of humic acid to HSA is the formation of HSA-humic acid complex with an association constant of 10 4 M -1 ; this corroborates the fluorescence lifetime measurements that the static mechanism was operated. The precise binding domain of humic acid in HSA has been verified from the denaturation of albumin, hydrophobic ANS displacement, and site-specific ligands; subdomain IIA (Sudlow's site I) was earmarked to possess high-affinity for humic acid. The observations are relevant for other albumin-humic substance systems when the ligands have analogous configuration with humic acid. - Highlights: → Albumin structure partially folds upon humic acid complexation. → Static type is dominance for the diminution in the Trp-214 fluorescence.→ Subdomain IIA is designate to possess high-affinity site for humic acid.

  9. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  10. The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

    Science.gov (United States)

    Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

    1996-05-03

    Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

  11. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    Science.gov (United States)

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  12. Genetic and biochemical identification of a novel single-stranded DNA binding complex in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Amy eStroud

    2012-06-01

    Full Text Available Single-stranded DNA binding proteins play an essential role in DNA replication and repair. They use oligosaccharide-binding folds, a five-stranded ß-sheet coiled into a closed barrel, to bind to single-stranded DNA thereby protecting and stabilizing the DNA. In eukaryotes the single-stranded DNA binding protein is known as replication protein A (RPA and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed single-stranded DNA-binding protein (SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3 exist in operons with a novel gene specific to Euryarchaeota, this gene encodes a protein that we have termed rpa-associated protein (RPAP. The rpap genes encode proteins belonging to COG3390 group and feature oligosaccharide-binding folds, suggesting that they might cooperate with RPA in binding to single-stranded DNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only ∆rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins. We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA binding complex that is unique to Euryarchaeota.

  13. Evaluation of DNA, BSA binding, and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline.

    Science.gov (United States)

    Moradi, Zohreh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam

    2018-02-01

    In order to evaluate biological potential of a novel synthesized complex [Nd(dmp) 2 Cl 3 .OH 2 ] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (K b ) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (K b  = 2.7 ± 0.07 × 10 5 ) and fluorescence spectroscopy (K b  = 1.13 ± 0.03 × 10 5 ). The Stern-Volmer constant (K SV ), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.

  14. Simple method for determining binding energies of fullerene and complex atomic negative ions

    Science.gov (United States)

    Felfli, Zineb; Msezane, Alfred

    2017-04-01

    A robust potential which embeds fully the vital core polarization interaction has been used in the Regge pole method to explore low-energy electron scattering from C60, Eu and Nb through the total cross sections (TCSs) calculations. From the characteristic dramatically sharp resonances in the TCSs manifesting negative ion formation in these systems, we extracted the binding energies for the C60, Euand Nbanions they are found to be in outstanding agreement with the measured electron affinities of C60, Eu and Nb. Common among these considered systems, including the standard atomic Au is the formation of their ground state negative ions at the second Ramsauer-Townsend (R-T) minima of their TCSs. Indeed, this is a signature of all the fullerenes and complex atoms considered thus far. Shape resonances, R-T minima and binding energies of the resultant anions are presented. This work was supported by U.S. DOE, Basic Energy Sciences, Office of Energy Research.

  15. MFIB: a repository of protein complexes with mutual folding induced by binding.

    Science.gov (United States)

    Fichó, Erzsébet; Reményi, István; Simon, István; Mészáros, Bálint

    2017-11-15

    It is commonplace that intrinsically disordered proteins (IDPs) are involved in crucial interactions in the living cell. However, the study of protein complexes formed exclusively by IDPs is hindered by the lack of data and such analyses remain sporadic. Systematic studies benefited other types of protein-protein interactions paving a way from basic science to therapeutics; yet these efforts require reliable datasets that are currently lacking for synergistically folding complexes of IDPs. Here we present the Mutual Folding Induced by Binding (MFIB) database, the first systematic collection of complexes formed exclusively by IDPs. MFIB contains an order of magnitude more data than any dataset used in corresponding studies and offers a wide coverage of known IDP complexes in terms of flexibility, oligomeric composition and protein function from all domains of life. The included complexes are grouped using a hierarchical classification and are complemented with structural and functional annotations. MFIB is backed by a firm development team and infrastructure, and together with possible future community collaboration it will provide the cornerstone for structural and functional studies of IDP complexes. MFIB is freely accessible at http://mfib.enzim.ttk.mta.hu/. The MFIB application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. simon.istvan@ttk.mta.hu, meszaros.balint@ttk.mta.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

  16. A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex

    DEFF Research Database (Denmark)

    Wiederhold, Katrin; Kloepper, Tobias H; Walter, Alexander M

    2010-01-01

    Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevi...

  17. Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Biswaranjan; Williams, Martin L.; Doak, Bradley C.; Vazirani, Mansha; Ilyichova, Olga [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); Wang, Geqing [La Trobe University, La Trobe Institute for Molecular Bioscience (Australia); Bermel, Wolfgang [Bruker Biospin GmbH (Germany); Simpson, Jamie S.; Chalmers, David K. [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); King, Glenn F. [The University of Queensland, Institute for Molecular Bioscience (Australia); Mobli, Mehdi, E-mail: m.mobli@uq.edu.au [The University of Queensland, Centre for Advanced Imaging (Australia); Scanlon, Martin J., E-mail: martin.scanlon@monash.edu [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia)

    2016-11-15

    We describe a general approach to determine the binding pose of small molecules in weakly bound protein–ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met {sup ε}CH{sub 3} assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-{sup 13}C,{sup 15}N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein–ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

  18. Mixed bilayer containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine: lipid complexation, ion binding, and electrostatics.

    Science.gov (United States)

    Pandit, Sagar A; Bostick, David; Berkowitz, Max L

    2003-11-01

    Two mixed bilayers containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine at a ratio of 5:1 are simulated in NaCl electrolyte solutions of different concentration using the molecular dynamics technique. Direct NH.O and CH.O hydrogen bonding between lipids was observed to serve as the basis of interlipid complexation. It is deduced from our results and previous studies that dipalmitoylphosphatidylcholine alone is less likely to form interlipid complexes than in the presence of bound ions or other bilayer "impurities" such as dipalmitoylphosphatidylserine. The binding of counterions is observed and quantitated. Based upon the calculated ion binding constants, the Gouy-Chapman surface potential (theta) is calculated. In addition we calculated the electrostatic potential profile (Phi) by twice integrating the system charge distribution. A large discrepancy between and the value of Phi at the membrane surface is observed. However, at "larger" distance from the bilayer surface, a qualitative similarity in the z-profiles of Phi and psi(GC) is seen. The discrepancy between the two potential profiles near the bilayer surface is attributed to the discrete and nonbulk-like nature of water in the interfacial region and to the complex geometry of this region.

  19. Synthesis and DNA binding/cleavage of mononuclear copper(II) phenanthroline/bipyridine proline complexes.

    Science.gov (United States)

    Reddy, Pulimamidi R; Raju, Nomula; Manjula, Pallerla; Reddy, Karnati V G

    2007-07-01

    The complexes [Cu(II)(phen)(L-Pro)(H2O)]+ ClO4(-) (1; phen = 1,10-phenanthroline) and [Cu(II)(bipy)(L-Pro)(H2O)]+ ClO4(-) (2; bipy = 2,2'-bipyridine) were synthesized and characterized by IR, magnetic susceptibility, UV/VIS, EPR, ESI-MS, elemental analysis, and theoretical calculations. The metal center was found in a square-pyramidal geometry. UV/VIS, thermal-denaturation, and fluorescence-spectroscopic studies were conducted to assess the interaction of the complexes with CT-DNA. An intercalative mode of binding was found, with intrinsic binding constants (Kb) of 3.86x10(3) and 4.6x10(3) M(-1) and Stern-Volmer quenching constants (K) of 0.15 and 0.11 for 1 and 2, respectively. Interestingly, none of the Cu(II) complexes was able to cleave pUC-19 DNA, which is attributed to the absence of a Pro amide H-atom and inhibition of the formation of an OH radical from the axially coordinated H2O molecule.

  20. Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET.

    Science.gov (United States)

    Fang, Bing; Zhang, Ming; Tian, Mai; Jiang, Lu; Guo, Hui Yuan; Ren, Fa Zheng

    2014-04-04

    α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF-OA was prepared and its activity and structural changes were compared with α-LA-OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF-OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF-OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA-OA. Similar to HAMLET, LF-OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF-OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein-OA complexes. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Preferential binding of yeast Rad4-Rad23 complex to damaged DNA

    International Nuclear Information System (INIS)

    Jansen, L.E.T.; Verhage, R.A.; Brouwer, J.

    1998-01-01

    The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER). Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER. We have purified an epitope-tagged Rad4.Rad23 (tRad4. Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus. tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER. Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro. UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex. These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage

  2. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    Science.gov (United States)

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  3. Characterization of the complex between native and reduced bovine serum albumin with aquacobalamin and evidence of dual tetrapyrrole binding.

    Science.gov (United States)

    Dereven'kov, Ilia A; Hannibal, Luciana; Makarov, Sergei V; Makarova, Anna S; Molodtsov, Pavel A; Koifman, Oskar I

    2018-05-02

    Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (H 2 OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H 2 OCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior to the formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the β-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H 2 OCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of H 2 OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.

  4. Interaction and Binding Modes of bis-Ruthenium(II Complex to Synthetic DNAs

    Directory of Open Access Journals (Sweden)

    Hasi Rani Barai

    2016-06-01

    Full Text Available [μ-(linkerL2(dipyrido[3,2-a:2′,3′-c]phenazine2(phenanthroline2Ru(II2]2+ with linker: 1,3-bis-(4-pyridyl-propane, L: PF6 (bis-Ru-bpp was synthesized and their binding properties to a various polynucleotides were investigated by spectroscopy, including normal absorption, circular dichroism(CD, linear dichroism(LD, and luminescence techniques in this study. On binding to polynucleotides, the bis-Ru-bpp complex with poly[d(A-T2], and poly[d(I-C2] exhibited a negative LDr signal whose intensity was as large as that in the DNA absorption region, followed by a complicated LDr signal in the metal-to-ligand charge transfer region. Also, the emission intensity and equilibrium constant of the bis-Ru-bpp complex with poly[d(A-T2], and poly[d(I-C2] were enhanced. It was reported that both of dppz ligand of the bis-Ru-bpp complex intercalated between DNA base-pairs when bound to native, mixed sequence DNA. Observed spectral properties resemble to those observed for poly[d(A-T2] and poly[d(I-C2], led us to be concluded that both dppz ligands intercalate between alternated AT and IC bases-pairs In contrast when bis-Ru-bpp complex was bound to poly[d(G-C2], the magnitude of the LDr in the dppz absorption region, as well as the emission intensity, was half in comparison to that of bound to poly[d(A-T2], and poly[d(I-C2]. Therefore the spectral properties of the bis-Ru-bpp-poly[d(G-C2] complex suggested deviation from bis-intercalation model in the poly[d(G-C2] case. These results can be explained by a model whereby one of the dppz ligands is intercalated while the other is exposed to solvent or may exist near to phosphate. Also it is indicative that the amine group of guanine in the minor groove provides the steric hindrance for incoming intercalation binder and it also takes an important role in a difference in binding of bis-Ru-bpp bound to poly[d(A-T2] and poly[d(I-C2].

  5. Cu(II Complexes of Isoniazid Schiff Bases: DNA/BSA Binding and Cytotoxicity Studies on A549 Cell Line

    Directory of Open Access Journals (Sweden)

    Pulipaka Ramadevi

    2014-01-01

    Full Text Available A series of isonicotinoyl hydrazones have been synthesized via template method and were complexed to Cu(II. The ligands are coordinated to Cu(II ion through the enolic oxygen and azomethine nitrogen resulting in a square planar geometry. The CT-DNA and bovine serum albumin binding propensities of the compounds were determined spectrophotometrically, the results of which indicate good binding propensity of complexes to DNA and BSA with high binding constant values. Furthermore, the compounds have been investigated for their cytotoxicities on A549 human lung cancer cell. Also the mode of cell death was examined employing various staining techniques and was found to be apoptotic.

  6. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    Directory of Open Access Journals (Sweden)

    Logan M. Decker

    2017-04-01

    Full Text Available In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD, which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC. Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute of the perinuclear meiotic silencing complex (MSC, directly linking the two cellular factors.

  7. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands

    Energy Technology Data Exchange (ETDEWEB)

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy, E-mail: drcjbstar@gmail.com

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL{sup 1}), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL{sup 2}), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL{sup 3}), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity. - Highlights: • Synthesis of ruthenium(II) hydrazone complexes • Molecular structure of the ligands was elucidated by single crystal X-ray diffraction method. • The ligands and complexes interact with CT-DNA via intercalation. • The complexes possess significant antioxidant activity against DPPH, OH and NO radicals. • The complex 6 shows higher IC{sub 50} value than the other complexes against cancer cells.

  8. A mononuclear zinc(II) complex with piroxicam: Crystal structure, DNA- and BSA-binding studies; in vitro cell cytotoxicity and molecular modeling of oxicam complexes

    Science.gov (United States)

    Jannesari, Zahra; Hadadzadeh, Hassan; Amirghofran, Zahra; Simpson, Jim; Khayamian, Taghi; Maleki, Batool

    2015-02-01

    A new mononuclear Zn(II) complex, trans-[Zn(Pir)2(DMSO)2], where Pir- is 4-hydroxy-2-methyl-N-2-pyridyl-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide (piroxicam), has been synthesized and characterized. The crystal structure of the complex was obtained by the single crystal X-ray diffraction technique. The interaction of the complex with DNA and BSA was investigated. The complex interacts with FS-DNA by two binding modes, viz., electrostatic and groove binding (major and minor). The microenvironment and the secondary structure of BSA are changed in the presence of the complex. The anticancer effects of the seven complexes of oxicam family were also determined on the human K562 cell lines and the results showed reasonable cytotoxicities. The interactions of the oxicam complexes with BSA and DNA were modeled by molecular docking and molecular dynamic simulation methods.

  9. The First Example of a Nitrile Hydratase Model Complex that Reversibly Binds Nitriles

    Science.gov (United States)

    Shearer, Jason; Jackson, Henry L.; Schweitzer, Dirk; Rittenberg, Durrell K.; Leavy, Tanya M.; Kaminsky, Werner; Scarrow, Robert C.; Kovacs, Julie A.

    2015-01-01

    Nitrile hydratase (NHase) is an iron-containing metalloenzyme that converts nitriles to amides. The mechanism by which this biochemical reaction occurs is unknown. One mechanism that has been proposed involves nucleophilic attack of an Fe-bound nitrile by water (or hydroxide). Reported herein is a five-coordinate model compound ([FeIII(S2Me2N3(Et,Pr))]+) containing Fe(III) in an environment resembling that of NHase, which reversibly binds a variety of nitriles, alcohols, amines, and thiocyanate. XAS shows that five-coordinate [FeIII(S2Me2N3(Et,Pr))]+ reacts with both methanol and acetonitrile to afford a six-coordinate solvent-bound complex. Competitive binding studies demonstrate that MeCN preferentially binds over ROH, suggesting that nitriles would be capable of displacing the H2O coordinated to the iron site of NHase. Thermodynamic parameters were determined for acetonitrile (ΔH = −6.2(±0.2) kcal/mol, ΔS = −29.4(±0.8) eu), benzonitrile (−4.2(±0.6) kcal/mol, ΔS = −18(±3) eu), and pyridine (ΔH = −8(±1) kcal/mol, ΔS = −41(±6) eu) binding to [FeIII(S2Me2N3(Et,Pr))]+ using variable-temperature electronic absorption spectroscopy. Ligand exchange kinetics were examined for acetonitrile, iso-propylnitrile, benzonitrile, and 4-tert-butylpyridine using 13C NMR line-broadening analysis, at a variety of temperatures. Activation parameters for ligand exchange were determined to be ΔH‡ = 7.1(±0.8) kcal/mol, ΔS‡ = −10(±1) eu (acetonitrile), ΔH‡ = 5.4(±0.6) kcal/mol, ΔS‡ = −17(±2) eu (iso-propionitrile), ΔH‡ = 4.9(±0.8) kcal/mol, ΔS‡ = −20(±3) eu (benzonitrile), and ΔH‡ = 4.7(±1.4) kcal/mol ΔS‡ = −18(±2) eu (4-tert-butylpyridine). The thermodynamic parameters for pyridine binding to a related complex, [FeIII(S2Me2N3(Pr,Pr))]+ (ΔH = −5.9(±0.8) kcal/mol, ΔS = −24(±3) eu), are also reported, as well as kinetic parameters for 4-tert-butylpyridine exchange (ΔH‡ = 3.1(±0.8) kcal/mol, ΔS‡)−25(±3) eu

  10. Immunoreactivity for calcium-binding proteins defines subregions of the vestibular nuclear complex of the cat.

    Science.gov (United States)

    Baizer, Joan S; Baker, James F

    2005-07-01

    The vestibular nuclear complex (VNC) is classically divided into four nuclei on the basis of cytoarchitectonics. However, anatomical data on the distribution of afferents to the VNC and the distribution of cells of origin of different efferent pathways suggest a more complex internal organization. Immunoreactivity for calcium-binding proteins has proven useful in many areas of the brain for revealing structure not visible with cell, fiber or Golgi stains. We have looked at the VNC of the cat using immunoreactivity for the calcium-binding proteins calbindin, calretinin and parvalbumin. Immunoreactivity for calretinin revealed a small, intensely stained region of cell bodies and processes just beneath the fourth ventricle in the medial vestibular nucleus. A presumably homologous region has been described in rodents. The calretinin-immunoreactive cells in this region were also immunoreactive for choline acetyltransferase. Evidence from other studies suggests that the calretinin region contributes to pathways involved in eye movement modulation but not generation. There were focal dense regions of fibers immunoreactive to calbindin in the medial and inferior nuclei, with an especially dense region of label at the border of the medial nucleus and the nucleus prepositus hypoglossi. There is anatomical evidence that suggests that the likely source of these calbindin-immunoreactive fibers is the flocculus of the cerebellum. The distribution of calbindin-immunoreactive fibers in the lateral and superior nuclei was much more uniform. Immunoreactivity to parvalbumin was widespread in fibers distributed throughout the VNC. The results suggest that neurochemical techniques may help to reveal the internal complexity in VNC organization.

  11. Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

    Science.gov (United States)

    Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

    1989-03-01

    The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

  12. Synthesis, characterization, DNA binding and catalytic applications of Ru(III) complexes.

    Science.gov (United States)

    Shoair, A F; El-Shobaky, A R; Azab, E A

    2015-01-01

    A new series of azodye ligands 5-chloro-3-hydroxy-4-(aryldiazenyl)pyridin-2(1H)-one (HLn) were synthesized by coupling of 5-chloro-3-hydroxypyridin-2(1H)-one with aniline and its p-derivatives. These ligands and their Ru(III) complexes of the type trans-[Ru(Ln)2(AsPh3)2]Cl were characterized by elemental analyses, IR, (1)H NMR and UV-Visible spectra as well as magnetic and thermal measurements. The molar conductance measurements proved that all the complexes are electrolytes. IR spectra show that the ligands (HLn) acts as a monobasic bidentate ligand by coordinating via the nitrogen atom of the azo group (NN) and oxygen atom of the deprotonated phenolic OH group, thereby forming a six-membered chelating ring and concomitant formation of an intramolecular hydrogen bond. The molecular and electronic structures of the investigated compounds (HLn) were also studied using quantum chemical calculations. The calf thymus DNA binding activity of the ligands (HLn) and their Ru(III) complexes were studied by absorption spectra and viscosity measurements. The mechanism and the catalytic oxidation of benzyl alcohol by trans-[Ru(Ln)2(AsPh3)2]Cl with hydrogen peroxide as co-oxidant were described. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose......-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  14. Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

    Science.gov (United States)

    Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

    2010-05-01

    Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

  15. Post-translational modifications near the quinone binding site of mammalian complex I.

    Science.gov (United States)

    Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2013-08-23

    Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

  16. Biochemical investigation of yttrium(III) complex containing 1,10-phenanthroline: DNA binding and antibacterial activity.

    Science.gov (United States)

    Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Moodi, Asieh; Niroomand, Sona

    2013-03-05

    Characterization of the interaction between yttrium(III) complex containing 1,10-phenanthroline as ligand, [Y(phen)2Cl(OH2)3]Cl2⋅H2O, and DNA has been carried out by UV absorption, fluorescence spectra and viscosity measurements in order to investigate binding mode. The experimental results indicate that the yttrium(III) complex binds to DNA and absorption is decreasing in charge transfer band with the increase in amount of DNA. The binding constant (Kb) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°), were calculated according to relevant fluorescent data and Vant' Hoff equation. The results of interaction mechanism studies, suggested that groove binding plays a major role in the binding of the complex and DNA. The activity of yttrium(III) complex against some bacteria was tested and antimicrobial screening tests shown growth inhibitory activity in the presence of yttrium(III) complex. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Kinetics and thermodynamics of small molecule binding to pincer-PCP rhodium(I) complexes

    KAUST Repository

    Doherty, Mark D.

    2013-04-15

    The kinetics and thermodynamics of the binding of several small molecules, L (L = N2, H2, D2, and C2H 4), to the coordinatively unsaturated pincer-PCP rhodium(I) complexes Rh[tBu2PCH2(C6H3)CH 2PtBu2] (1) and Rh[tBu 2P(CH2)2(CH)(CH2)2P tBu2] (2) in organic solvents (n-heptane, toluene, THF, and cyclohexane-d12) have been investigated by a combination of kinetic flash photolysis methods, NMR equilibrium studies, and density functional theory (DFT) calculations. Using various gas mixtures and monitoring by NMR until equilibrium was established, the relative free energies of binding of N2, H2, and C2H4 in cyclohexane-d12 were found to increase in the order C 2H4 < N2 < H2. Time-resolved infrared (TRIR) and UV-vis transient absorption spectroscopy revealed that 355 nm excitation of 1-L and 2-L results in the photoejection of ligand L. The subsequent mechanism of binding of L to 1 and 2 to regenerate 1-L and 2-L is determined by the structure of the PCP ligand framework and the nature of the solvent. In both cases, the primary transient is a long-lived, unsolvated species (τ = 50-800 ns, depending on L and its concentration in solution). For 2, this so-called less-reactive form (LRF) is in equilibrium with a more-reactive form (MRF), which reacts with L at diffusion-controlled rates to regenerate 2-L. These two intermediates are proposed to be different conformers of the three-coordinate (PCP)Rh fragment. For 1, a similar mechanism is proposed to occur, but the LRF to MRF step is irreversible. In addition, a parallel reaction pathway was observed that involves the direct reaction of the LRF of 1 with L, with second-order rate constants that vary by almost 3 orders of magnitude, depending on the nature of L (in n-heptane, k = 6.7 × 10 5 M-1 s-1 for L = C2H4; 4.0 × 106 M-1 s-1 for L = N2; 5.5 × 108 M-1 s-1 for L = H2). Experiments in the more coordinating solvent, THF, revealed the binding of THF to 1 to generate 1-THF, and its subsequent reaction with L, as a

  18. Kinetics and thermodynamics of small molecule binding to pincer-PCP rhodium(I) complexes

    KAUST Repository

    Doherty, Mark D.; Grills, David C.; Huang, Kuo-Wei; Muckerman, James T.; Polyansky, Dmitry E.; Van Eldik, Rudi V.; Fujita, Etsuko

    2013-01-01

    The kinetics and thermodynamics of the binding of several small molecules, L (L = N2, H2, D2, and C2H 4), to the coordinatively unsaturated pincer-PCP rhodium(I) complexes Rh[tBu2PCH2(C6H3)CH 2PtBu2] (1) and Rh[tBu 2P(CH2)2(CH)(CH2)2P tBu2] (2) in organic solvents (n-heptane, toluene, THF, and cyclohexane-d12) have been investigated by a combination of kinetic flash photolysis methods, NMR equilibrium studies, and density functional theory (DFT) calculations. Using various gas mixtures and monitoring by NMR until equilibrium was established, the relative free energies of binding of N2, H2, and C2H4 in cyclohexane-d12 were found to increase in the order C 2H4 < N2 < H2. Time-resolved infrared (TRIR) and UV-vis transient absorption spectroscopy revealed that 355 nm excitation of 1-L and 2-L results in the photoejection of ligand L. The subsequent mechanism of binding of L to 1 and 2 to regenerate 1-L and 2-L is determined by the structure of the PCP ligand framework and the nature of the solvent. In both cases, the primary transient is a long-lived, unsolvated species (τ = 50-800 ns, depending on L and its concentration in solution). For 2, this so-called less-reactive form (LRF) is in equilibrium with a more-reactive form (MRF), which reacts with L at diffusion-controlled rates to regenerate 2-L. These two intermediates are proposed to be different conformers of the three-coordinate (PCP)Rh fragment. For 1, a similar mechanism is proposed to occur, but the LRF to MRF step is irreversible. In addition, a parallel reaction pathway was observed that involves the direct reaction of the LRF of 1 with L, with second-order rate constants that vary by almost 3 orders of magnitude, depending on the nature of L (in n-heptane, k = 6.7 × 10 5 M-1 s-1 for L = C2H4; 4.0 × 106 M-1 s-1 for L = N2; 5.5 × 108 M-1 s-1 for L = H2). Experiments in the more coordinating solvent, THF, revealed the binding of THF to 1 to generate 1-THF, and its subsequent reaction with L, as a

  19. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    OpenAIRE

    Qin, Yangzhong; Yang, Yi; Zhang, Luyuan; Fowler, Jason D.; Qiu, Weihong; Wang, Lijuan; Suo, Zucai; Zhong, Dongping

    2013-01-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and terna...

  20. Synthesis, structure, DNA binding and anticancer activity of mixed ligand ruthenium(II) complex

    Science.gov (United States)

    Gilewska, Agnieszka; Masternak, Joanna; Kazimierczuk, Katarzyna; Trynda, Justyna; Wietrzyk, Joanna; Barszcz, Barbara

    2018-03-01

    In order to obtain a potential chemotherapeutic which is not affected on the normal BALB/3T3 cell line, a new arene ruthenium(II) complex {[RuCl(L1)(η6-p-cymene)]PF6}2 · H2O has been synthesized by a direct reaction of precursor, [{(η6-p-cymene)Ru(μ-Cl)}2Cl2], with N,N-chelating ligand (L1 - 2,2‧-bis(4,5-dimethylimidazole). The compound has been fully characterized by elemental analysis, X-ray diffraction, IR, UV-Vis and 1H, 13C NMR spectroscopies. X-ray analysis have confirmed that the compound crystallized in the monoclinic group Cc as an inversion twin. The asymmetric unit contains two symmetrically independent cationic complexes [RuCl(L1)(η6-p-cymene)]+ whose charge is balanced by two PF6- counterions. The shape of each cationic coordination polyhedral can be described as a distorted dodecahedron and shows a typical piano-stool geometry. In addition, an analysis of the crystal structure and the Hirshfeld surface analysis were used to detect and visualize important hydrogen bonds and intermolecular interaction. Moreover, the antiproliferative behavior of the obtained complex was assayed against three human cells: MV-4-11, LoVo, MCF-7 and BALB/3T3 - normal mice fibroblast cells. To predict a binding mode, a potential interaction of ruthenium complex with calf thymus DNA (CT-DNA) has been explored using UV absorption and circular dichroism (CD).

  1. Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

    Science.gov (United States)

    Yoneyama, Tohru; Miyata, Keita; Chikai, Tomoyuki; Mikami, Akifumi; Suzuki, Tomonori; Hasegawa, Kimiko; Ikeda, Toshihiko; Watanabe, Toshihiro; Ohyama, Tohru; Niwa, Koichi

    2008-12-01

    Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

  2. Apparent partition coefficient in octanol-water and binding percentage to BSA of 153Sm(113,117Snm) complexes

    International Nuclear Information System (INIS)

    Yang Yuqing; Luo Shunzhong; Wang Guanquan; He Jiaheng; Bing Wenzeng; Pu Manfei; Wei Hongyuan; Wang Wenjin

    2004-01-01

    Apparent partition coefficient in octanol-water and binding percentage to BSA of 153 Sm-NTMP, 153 Sm-HEDTMP, 153 Sm-DCTMP, 153 Sm-EDTMP, 153 Sm-DTPMP, 113,117 Sn m -EDTMP, 113,117 Sn m -HEDTMP, 113,117 Sn m -DTPMP are measured. The results show that there is a linear relationship between the relative magnitude of the apparent partition coefficient in octanol-water and the relative magnitude of the binding percentage to BSA of these 153 Sm( 113,117 Sn m ) complexes. This linear relationship provides a new method for determination of the apparent partition coefficient in octanol-water of 153 Sm( 113,117 Sn m ) complexes of this kind. This linear relationship also implicates that hydrophobic force plays an important role in the binding of 153 Sm( 113,117 Sn m ) complexes to BSA

  3. A Rac1--GDP trimer complex binds zinc with tetrahedral and octahedral coordination, displacing magnesium

    Energy Technology Data Exchange (ETDEWEB)

    Prehna, G.; Stebbins, C

    2007-01-01

    The Rho family of small GTPases represent well characterized signaling molecules that regulate many cellular functions such as actin cytoskeletal arrangement and the cell cycle by acting as molecular switches. A Rac1-GDP-Zn complex has been crystallized in space group P3221 and its crystal structure has been solved at 1.9 {angstrom} resolution. These trigonal crystals reveal the unexpected ability of Rac1 to coordinate Zn atoms in a tetrahedral fashion by use of its biologically relevant switch I and switch II regions. Upon coordination of zinc, the switch I region is stabilized in the GDP-bound conformation and contributes to a Rac1 trimer in the asymmetric unit. Zinc coordination causes switch II to adopt a novel conformation with a symmetry-related molecule. Additionally, zinc was found to displace magnesium from its octahedral coordination at switch I, although GDP binding remained stable. This structure represents the first reported Rac1-GDP-Zn complex, which further underscores the conformational flexibility and versatility of the small GTPase switch regions.

  4. A Rac1-GDP Trimer Complex Binds Zinc with Tetrahedral and Octahedral Coordination, Displacing Magnesium

    Energy Technology Data Exchange (ETDEWEB)

    Prehna,G.; Stebbins, E.

    2007-01-01

    The Rho family of small GTPases represent well characterized signaling molecules that regulate many cellular functions such as actin cytoskeletal arrangement and the cell cycle by acting as molecular switches. A Rac1-GDP-Zn complex has been crystallized in space group P3{sub 2}21 and its crystal structure has been solved at 1.9 {angstrom} resolution. These trigonal crystals reveal the unexpected ability of Rac1 to coordinate Zn atoms in a tetrahedral fashion by use of its biologically relevant switch I and switch II regions. Upon coordination of zinc, the switch I region is stabilized in the GDP-bound conformation and contributes to a Rac1 trimer in the asymmetric unit. Zinc coordination causes switch II to adopt a novel conformation with a symmetry-related molecule. Additionally, zinc was found to displace magnesium from its octahedral coordination at switch I, although GDP binding remained stable. This structure represents the first reported Rac1-GDP-Zn complex, which further underscores the conformational flexibility and versatility of the small GTPase switch regions.

  5. Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

    Science.gov (United States)

    Swift, Kerry M.; Matayoshi, Edmund D.

    2008-02-01

    FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

  6. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    DEFF Research Database (Denmark)

    Nielsen, C H; Svehag, S E; Marquart, H V

    1994-01-01

    binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC-induced degranulation...

  7. CLINICAL EXPERIENCE OF CANCER IMMUNOTHERAPY INTEGRATED WITH OLEIC ACID COMPLEXED WITH DE-GLYCOSYLATED VITAMIN D BINDING PROTEIN

    OpenAIRE

    Emma Ward; Rodney Smith; Jacopo J.V. Branca; David Noakes; Gabriele Morucci; Lynda Thyer

    2014-01-01

    Proteins highly represented in milk such as α-lactalbumin and lactoferrin bind Oleic Acid (OA) to form complexes with selective anti-tumor activity. A protein present in milk, colostrum and blood, vitamin D binding protein is the precursor of a potent Macrophage Activating Factor (GcMAF) and in analogy with other OA-protein complexes, we proposed that OA-GcMAF could demonstrate a greater immunotherapeutic activity than that of GcMAF alone. We describe a preliminary experience treating p...

  8. Binding of α2-macroglobulin-thrombin complexes and methylamine-treated α2-macroglobulin to human blood monocytes

    International Nuclear Information System (INIS)

    Straight, D.L.; Jakoi, L.; McKee, P.A.; Snyderman, R.

    1988-01-01

    The binding of α 2 -macroglobulin (α 2 M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125 I-labeled α 2 M. Cells and bound ligand were separated from free ligand by rapid vacuum filtration. Nonlinear least-squares analysis of data obtained in direct binding studies at 0 0 C showed that monocytes bound the α 2 M-thrombin complex with a K/sub d/ 3.0 +- .09 nM and the monocyte had 1545 +- 153 sitescell. Thrombin alone did not compete for the site. Binding was divalent cation dependent. Direct binding studies also demonstrated that monocytes bound methylamine-treated α 2 M in a manner similar to α 2 M-thrombin. Competitive binding studies showed that α 2 M-thrombin and methylamine-treated α 2 M bound to the same sites on the monocyte. In contrast, native α 2 M did not compete with α 2 M-thrombin for the site. Studies done at 37 0 C suggested that after binding, the monocyte internalized and degraded α 2 M-thrombin and excreted the degradation products. Receptor turnover and degradation of α 2 M-thrombin complexes were blocked in monocytes treated with chloroquine, an inhibitor of lysosomal function. The results indicate that human monocytes have a divalent cation dependent, high-affinity binding site for α 2 M-thrombin and methylamine-treated α 2 M which may function to clear α 2 M-proteinase complexes from the circulation

  9. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  10. Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

    Science.gov (United States)

    Wise, Jillian F; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K; Tao, Rong-Hua; Sabichi, Anita L; Ao, Xue; Maeng, Hoyoung; Samaniego, Felipe

    2013-06-06

    Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

  11. Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: a tight complex implying one binding site.

    Science.gov (United States)

    Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

    2012-10-01

    The intrinsically disordered C-terminal domain (N(TAIL) ) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The N(TAIL) region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489-506. In the previous studies published in this journal, we obtained experimental evidence supporting a K(D) for the N(TAIL) -XD binding reaction in the nM range and also showed that an additional N(TAIL) region (Box3, aa 517-525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K(D) in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in N(TAIL) -XD binding. Since our previous studies relied on N(TAIL) -truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an N(TAIL) devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482-525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N(TAIL) forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. Copyright © 2012 The Protein Society.

  12. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  13. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  14. Mechanistic Aspects of the Reversible Binding of SO2 on Arylplatinum Complexes: Experimental and ab Initio Studies

    NARCIS (Netherlands)

    Koten, G. van; Albrecht, M.A.; Gossage, R.A.; Frey, H.; Ehlers, A.W.; Baerends, E.J.; Merbach, A.E.

    2001-01-01

    The detailed mechanism of the reversible binding and fast exchange of SO2 on the organoplatinum(II) complex [PtI(NCN)], 1, has been studied experimentally in solution (C2F4Br2) using low-temperature NMR spectroscopy and theoretically by ab initio calculations. Direct bonding of SO2 and formation of

  15. Mononuclear Pd(II) complex as a new therapeutic agent: Synthesis, characterization, biological activity, spectral and DNA binding approaches

    Science.gov (United States)

    Saeidifar, Maryam; Mirzaei, Hamidreza; Ahmadi Nasab, Navid; Mansouri-Torshizi, Hassan

    2017-11-01

    The binding ability between a new water-soluble palladium(II) complex [Pd(bpy)(bez-dtc)]Cl (where bpy is 2,2‧-bipyridine and bez-dtc is benzyl dithiocarbamate), as an antitumor agent, and calf thymus DNA was evaluated using various physicochemical methods, such as UV-Vis absorption, Competitive fluorescence studies, viscosity measurement, zeta potential and circular dichroism (CD) spectroscopy. The Pd(II) complex was synthesized and characterized using elemental analysis, molar conductivity measurements, FT-IR, 1H NMR, 13C NMR and electronic spectra studies. The anticancer activity against HeLa cell lines demonstrated lower cytotoxicity than cisplatin. The binding constants and the thermodynamic parameters were determined at different temperatures (300 K, 310 K and 320 K) and shown that the complex can bind to DNA via electrostatic forces. Furthermore, this result was confirmed by the viscosity and zeta potential measurements. The CD spectral results demonstrated that the binding of Pd(II) complex to DNA induced conformational changes in DNA. We hope that these results will provide a basis for further studies and practical clinical use of anticancer drugs.

  16. A Polypyrimidine Tract Binding Protein, Pumpkin RBP50, Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex[W

    Science.gov (United States)

    Ham, Byung-Kook; Brandom, Jeri L.; Xoconostle-Cázares, Beatriz; Ringgold, Vanessa; Lough, Tony J.; Lucas, William J.

    2009-01-01

    RNA binding proteins (RBPs) are integral components of ribonucleoprotein (RNP) complexes and play a central role in RNA processing. In plants, some RBPs function in a non-cell-autonomous manner. The angiosperm phloem translocation stream contains a unique population of RBPs, but little is known regarding the nature of the proteins and mRNA species that constitute phloem-mobile RNP complexes. Here, we identified and characterized a 50-kD pumpkin (Cucurbita maxima cv Big Max) phloem RNA binding protein (RBP50) that is evolutionarily related to animal polypyrimidine tract binding proteins. In situ hybridization studies indicated a high level of RBP50 transcripts in companion cells, while immunolocalization experiments detected RBP50 in both companion cells and sieve elements. A comparison of the levels of RBP50 present in vascular bundles and phloem sap indicated that this protein is highly enriched in the phloem sap. Heterografting experiments confirmed that RBP50 is translocated from source to sink tissues. Collectively, these findings established that RBP50 functions as a non-cell-autonomous RBP. Protein overlay, coimmunoprecipitation, and cross-linking experiments identified the phloem proteins and mRNA species that constitute RBP50-based RNP complexes. Gel mobility-shift assays demonstrated that specificity, with respect to the bound mRNA, is established by the polypyrimidine tract binding motifs within such transcripts. We present a model for RBP50-based RNP complexes within the pumpkin phloem translocation stream. PMID:19122103

  17. Competitive binding affinity of two lanthanum(III) macrocycle complexes toward DNA and bovine serum albumin in water

    Energy Technology Data Exchange (ETDEWEB)

    Asadi, Zahra; Mosallaei, Hamta; Sedaghat, Moslem [Shiraz Univ. (Iran, Islamic Republic of). Dept. of Chemistry; Yousefi, Reza [Shiraz Univ. (Iran, Islamic Republic of). Protein Chemistry Lab. (PCL)

    2017-11-15

    In the present study, two water-soluble lanthanum(III) hexaaza Schiff base complexes were synthesized and characterized and also theoretically investigated. The interactions of these complexes with DNA and bovine serum albumin (BSA) were studied using different spectroscopic assessments and docking simulation analysis. The DNA docking studies suggested that these two complexes are able to interact with DNA through the minor groove, and also the binding affinity is in the order of La(L{sup 1}) > La(L{sup 2}). Furthermore, the spectral titration was carried out and viscosity measurements were taken. In this regard, protein-binding studies revealed that these complexes quench the intrinsic fluorescence of BSA, and indicated that the possible binding site is located on the vicinity of Trp 213, which is further validated by docking simulation analysis. The in vitro anticancer activities of these complexes indicated that the La(L{sup 1}) complex is more effective than the other one and also exhibits a better interaction with DNA.

  18. ct-DNA Binding and Antibacterial Activity of Octahedral Titanium (IV Heteroleptic (Benzoylacetone and Hydroxamic Acids Complexes

    Directory of Open Access Journals (Sweden)

    Raj Kaushal

    2016-01-01

    Full Text Available Five structurally related titanium (IV heteroleptic complexes, [TiCl2(bzac(L1–4] and [TiCl3(bzac(HL5]; bzac = benzoylacetonate; L1–5 = benzohydroximate (L1, salicylhydroximate (L2, acetohydroximate (L3, hydroxyurea (L4, and N-benzoyl-N-phenyl hydroxylamine (L5, were used for the assessment of their antibacterial activities against ten pathogenic bacterial strains. The titanium (IV complexes (1–5 demonstrated significant level of antibacterial properties as measured using agar well diffusion method. UV-Vis absorption spectroscopic technique was applied, to get a better insight into the nature of binding between titanium (IV complexes with calf thymus DNA (ct-DNA. On the basis of the results of UV-Vis absorption spectroscopy, the interaction between ct-DNA and the titanium (IV complexes is likely to occur through the same mode. Results indicated that titanium (IV complex can bind to calf thymus DNA (ct-DNA via an intercalative mode. The intrinsic binding constant (Kb was calculated by absorption spectra by using Benesi-Hildebrand equation. Further, Gibbs free energy was also calculated for all the complexes.

  19. Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

    Science.gov (United States)

    Vorobyeva, Nadezhda E.; Mazina, Marina U.; Golovnin, Anton K.; Kopytova, Daria V.; Gurskiy, Dmitriy Y.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Georgiev, Pavel G.; Krasnov, Aleksey N.

    2013-01-01

    Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein. PMID:23609538

  20. Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis

    Science.gov (United States)

    Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

    2016-09-01

    The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb = (7.6 ± 0.21) × 105) between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2 ± 0.11) × 106 M- 1. Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

  1. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    DEFF Research Database (Denmark)

    Nielsen, C H; Svehag, S E; Marquart, H V

    1994-01-01

    -binding to granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min...... to the findings for PMN, the difference between IC-binding to monocytes in the absence and presence of E increased progressively over the 90 min observation period, suggesting that different mechanisms are involved in the late-phase IC uptake by monocytes and PMN. Lymphocytes were heterogeneous with respect to IC...... binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC-induced degranulation...

  2. Detection of immune complexes in sera of dogs with rheumatic and neoplastic diseases by 125I-Clq binding test

    International Nuclear Information System (INIS)

    Terman, D.S.; Moore, D.; Collins, J.; Johnston, B.; Person, D.; Templeton, J.; Poser, R.; Quinby, F.

    1979-01-01

    Some canine rheumatic and neoplastic diseases bear a striking clinical and serological resemblance to their counterparts in man. In the present study, human 125 I-Clq was employed in a radioimmunoassay for detection of immune complexes in sera of normal dogs and those with rheumatic and neoplastic diseases. Human 125 I-Clq showed binding of 16.7 +- 5.73% in a group of normal dog sera with binding of 32.5 +- 17.3% and 43.0 +- 16.0% in sera of dogs with rheumatic and neoplastic diseases. respectively. Human 125 I-Clq bound similar quantities of heat-aggregated canine and human gamma-globulin over a broad range of concentrations and human 125 I-Clq binding in canine sera was effectively inhibited by similar quantities of heat aggregated canine and human gamma-globulin. Seven of 12 dogs with elevated levels of Clq binding had active clinical and serological rheumatic disease (SLE or rheumatoid arthritis), while none of 7 dogs with values within the normal range had active clinical disease. All 5 dogs with widespread osteogenic sarcoma and all 4 dogs with high grade adenocarcinoma of the mammary gland had elevated Clq binding values while 2 animals with low grade malignancies without evident metastases did not. Thus, it appears that human 125 I-Clq may be employed to assay immune complexes in canine sera and may be a valuable technique for the study of dogs with various rheumatic and neoplastic diseases. (author)

  3. A monofunctional platinum complex coordinated to a rhodium metalloinsertor selectively binds mismatched DNA in the minor groove.

    Science.gov (United States)

    Weidmann, Alyson G; Barton, Jacqueline K

    2015-10-05

    We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

  4. Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

    DEFF Research Database (Denmark)

    Meier, Sebastian; Beeren, Sophie

    2014-01-01

    We describe a simple method for the simultaneous determination of association constants for a guest binding to seven different hosts in a mixture of more than 20 different oligosaccharides. If the binding parameters are known for one component in the mixture, a single NMR titration suffices...

  5. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug-binding site

    OpenAIRE

    Handing, Katarzyna B.; Shabalin, Ivan G.; Szlachta, Karol; Majorek, Karolina A.; Minor, Wladek

    2016-01-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1 ?. Cetirizine is bound in two sites ? a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizi...

  6. The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β2-Microglobulin through Distinct Binding Sites

    DEFF Research Database (Denmark)

    Merkle, Patrick S.; Irving, Melita; Hongjian, Song

    2017-01-01

    from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and......T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics...... are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR...

  7. Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

    Directory of Open Access Journals (Sweden)

    Rosner William

    2009-05-01

    Full Text Available Abstract Background Human sex hormone-binding globulin (SHBG regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (PL. The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (PT, and two minor transcripts. Results We report that transcriptional expression of the human SHBG gene is far more complex than previously described. PL and PT direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of PL and PT, and present evidence for a third SHBG gene promoter (PN within the neighboring FXR2 gene; PN regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N. Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. PL- PT- and PN-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells. Conclusion These results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

  8. A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Thorgersen, Michael P. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Lancaster, W. Andrew [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Rajeev, Lara [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Ge, Xiaoxuan [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Vaccaro, Brian J. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Poole, Farris L. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Arkin, Adam P. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mukhopadhyay, Aindrila [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Adams, Michael W. W. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology

    2016-12-02

    Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by

  9. Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

    Science.gov (United States)

    Kramers, C; Hylkema, M N; van Bruggen, M C; van de Lagemaat, R; Dijkman, H B; Assmann, K J; Smeenk, R J; Berden, J H

    1994-01-01

    Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. Images PMID:8040312

  10. Serum albumin forms a lactoferrin-like soluble iron-binding complex in presence of hydrogen carbonate ions.

    Science.gov (United States)

    Ueno, Hiroshi M; Urazono, Hiroshi; Kobayashi, Toshiya

    2014-02-15

    The iron-lactoferrin complex is a common food ingredient because of its iron-solubilizing capability in the presence of hydrogen carbonate ions. However, it is unclear whether the formation of a stable iron-binding complex is limited to lactoferrin. In this study, we investigated the effects of bovine serum albumin (BSA) on iron solubility and iron-catalyzed lipid oxidation in the presence of hydrogen carbonate ions. BSA could solubilize >100-fold molar equivalents of iron at neutral pH, exceeding the specific metal-binding property of BSA. This iron-solubilizing capability of BSA was impaired by thermally denaturing BSA at ≥ 70 °C for 10 min at pH 8.5. The resulting iron-BSA complex inhibited iron-catalyzed oxidation of soybean oil in a water-in-oil emulsion measured using the Rancimat test. Our study is the first to show that BSA, like lactoferrin, forms a soluble iron-binding complex in the presence of hydrogen carbonate ions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Computational prediction of binding affinity for CYP1A2-ligand complexes using empirical free energy calculations

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Olsen, Lars; Jørgensen, Flemming Steen

    2010-01-01

    , and methods based on statistical mechanics. In the present investigation, we started from an LIE model to predict the binding free energy of structurally diverse compounds of cytochrome P450 1A2 ligands, one of the important human metabolizing isoforms of the cytochrome P450 family. The data set includes both...... substrates and inhibitors. It appears that the electrostatic contribution to the binding free energy becomes negligible in this particular protein and a simple empirical model was derived, based on a training set of eight compounds. The root mean square error for the training set was 3.7 kJ/mol. Subsequent......Predicting binding affinities for receptor-ligand complexes is still one of the challenging processes in computational structure-based ligand design. Many computational methods have been developed to achieve this goal, such as docking and scoring methods, the linear interaction energy (LIE) method...

  12. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    DEFF Research Database (Denmark)

    Dou, Q P; Zhao, S; Levin, A H

    1994-01-01

    report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes...... complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex...

  13. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    International Nuclear Information System (INIS)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-01-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4. (paper)

  14. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    Science.gov (United States)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-10-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

  15. PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

    Science.gov (United States)

    McGuire, V; Alexander, S

    1996-06-14

    The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

  16. Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

    International Nuclear Information System (INIS)

    Jaru-ampornpan, Peera; Narkpuk, Jaraspim; Wanitchang, Asawin; Jongkaewwattana, Anan

    2014-01-01

    Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference

  17. Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Jaru-ampornpan, Peera, E-mail: peera.jar@biotec.or.th; Narkpuk, Jaraspim; Wanitchang, Asawin; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2014-01-03

    Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.

  18. Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein*

    Science.gov (United States)

    Grüning, Clara S. R.; Mirecka, Ewa A.; Klein, Antonia N.; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F.; Stoldt, Matthias; Hoyer, Wolfgang

    2014-01-01

    The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. PMID:24966331

  19. Inclusion complex formation of ternary system: Fluoroscein-p-sulfonato calix[4]arene-Cu(2+) by cooperative binding.

    Science.gov (United States)

    Gawhale, Sharadchandra; Jadhav, Ankita; Rathod, Nilesh; Malkhede, Dipalee; Chaudhari, Gajanan

    2015-09-05

    The aqueous solution of fluorescein-para sulfonato calix[4]arene-metal ion complex has been studied based on absorption, fluorescence, (1)H NMR and FTIR spectroscopic results. It was found that the fluorescence intensity quenched regularly upon addition of pSCX4 and metal ion. The quenching constants and binding constants were determined for pSCX4-FL and pSCX4-FL-Cu(2+) systems. 1:1 stoichiometry is obtained for pSCX4-Cu(2+) system by continuous variation method. The NMR and IR results indicates the interaction among FL, pSCX4 and Cu(2+). The combined results demonstrate the cooperative binding to design the complex for ternary system. The life time for binary and ternary system has been studied. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug-binding peptide.

    Science.gov (United States)

    Gocheva, Gergana; Ilieva, Nina; Peneva, Kalina; Ivanova, Anela

    2018-04-01

    Polypeptide-based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular-level approach is adopted for investigating the non-covalent interactions between doxorubicin and a recently synthesized drug-binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug-peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug-peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π-Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra- and intermolecular H-bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug-to-peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin-binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems. © 2017 John Wiley & Sons A/S.

  1. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  2. A study of core electron binding energies in technetium-99m complexes by internal conversion electron spectroscopy

    International Nuclear Information System (INIS)

    Burke, J.F.; Archer, C.M.; Wei Chiu, K.; Latham, I.A.; Egdell, R.G.

    1991-01-01

    Core electron binding energies in a series of 99m Tc complexes have been studied by internal conversion electron spectroscopy (ICES) in a conventional x-ray photoelectron spectrometer. In both 3d and 3p regions, a chemical shift of about 1 eV is observed per unit increase in oxidation state. The role of ICES in characterizing radiopharmaceutical agents is illustrated with studies of some novel 99m Tc-phosphine complexes that have been developed for myocardial perfusion imaging. (author)

  3. A Bulky Rhodium Complex Bound to an Adenosine-Adenosine DNA Mismatch: General Architecture of the Metalloinsertion Binding Mode†

    Science.gov (United States)

    Zeglis, Brian M.; Pierre, Valérie C.; Kaiser, Jens T.; Barton, Jacqueline K.

    2009-01-01

    Two crystal structures are determined for Δ-Rh(bpy)2(chrysi)3+ (chrysi = 5,6-chrysenequinone diimine) bound to the oligonucleotide duplex 5′-CGGAAATTACCG-3′ containing two adenosine-adenosine mismatches (italics) through metalloinsertion. Diffraction quality crystals with two different space groups (P3221 and P43212) were obtained under very similar crystallization conditions. In both structures, the bulky rhodium complex inserts into the two mismatched sites from the minor groove side, ejecting the mismatched bases into the major groove. The conformational changes are localized to the mismatched site; the metal complex replaces the mismatched base pair without an increase in base pair rise. The expansive metal complex is accommodated in the duplex by a slight opening in the phosphodiester backbone; all sugars retain a C2′-endo puckering, and flanking base pairs neither stretch nor shear. The structures differ, however, in that in one of the structures, an additional metal complex is bound by intercalation from the major groove at the central 5′-AT-3′ step. We conclude that this additional metal complex is intercalated into this central step because of crystal packing forces. The structures described here of Δ-Rh(bpy)2(chrysi)3+ bound to thermodynamically destabilized AA mismatches share critical features with binding by metalloinsertion in two other oligonucleotides containing different single base mismatches. These results underscore the generality of the metalloinsertion as a new mode of non-covalent binding by small molecules with a DNA duplex. PMID:19374348

  4. Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

    Science.gov (United States)

    Gul, Ahmet; Erman, Burak

    2018-03-01

    Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

  5. Investigation of the complex structure, comparative DNA-binding and DNA cleavage of two water-soluble mono-nuclear lanthanum(III) complexes and cytotoxic activity of chitosan-coated magnetic nanoparticles as drug delivery for the complexes

    Science.gov (United States)

    Asadi, Zahra; Nasrollahi, Neda; Karbalaei-Heidari, Hamidreza; Eigner, Vaclav; Dusek, Michal; Mobaraki, Nabiallah; Pournejati, Roya

    2017-05-01

    Two water-soluble mono-nuclear macrocyclic lanthanum(III) complexes of 2,6-diformyl-4-methylphenol with 1,3-diamino-2-propanol (C1) or 1,3-propylenediamine (C2) were synthesized and characterized by UV-Vis, FT-IR, 13C and 1H NMR spectroscopy and elemental analysis. C1 complex was structurally characterized by single-crystal X-ray diffraction, which revealed that the complex was mononuclear and ten-coordinated. The coordination sites around lanthanum(III) were occupied with a five-dentate ligand, two bidentate nitrates, and one water molecule. The interaction of complexes with DNA was studied in buffered aqueous solution at pH 7.4. UV-Vis absorption spectroscopy, emission spectroscopy, circular dichroism (CD) and viscometric measurements provided clear evidence of the intercalation mechanism of binding. The obtained intrinsic binding constants (Kb) 9.3 × 103 and 1.2 × 103 M- 1 for C1 and C2, respectively confirmed that C1 is better intercalator than C2. The DNA docking studies suggested that the complexes bind with DNA in a groove binding mode with the binding affinity of C1 > C2. Moreover, agarose gel electrophoresis study of the DNA-complex for both compounds revealed that the C1 intercalation cause ethidium bromide replacement in a competitive manner which confirms the suggested mechanism of binding. Finally, the anticancer experiments for the treated cancerous cell lines with both synthesized compounds show that these hydrophilic molecules need a suitable carrier to pass through the hydrophobic nature of cell membrane efficiently.

  6. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

    Science.gov (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

    1996-11-01

    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  7. Relationship of binding specificity and structural property of the technetium-99m complexes for tumor hypoxia and tumor angiogenesis imaging

    International Nuclear Information System (INIS)

    Su, Z.F.

    2005-01-01

    The growth of tumor requires nutrition and oxygen. Tumor cells will become hypoxic when the supply of oxygen is insufficient. Hypoxic tumor cells will not only resist radiation therapy and chemotherapy, but also induce angiogenesis for oxygen supply and for metastasis. Therefore, detection of tumor hypoxia and tumor angiogenesis with high sensitive radio labeled imaging agents is important. Hypoxic tumor cells may display some molecules as tumor markers for the specific binding with radiopharmaceuticals. Radiopharmaceuticals, unlike the non-radioactive drugs, are trace compounds in a given dosage. Due to the extreme low concentration, the non-specific accumulation of the radiotracers by blood cells and proteins, tissues, and organs can be even more serious compared to the non-radioactive drugs. The non-specific accumulation of the radiotracers can make the ratios of tumor/tissue (in terms of i.d.%/g) falling to the range of 2∼7 [1-2]. Non-specific binding of radiopharmaceuticals is common, but detailed studies on it are poor documented. This presentation reports the study of the relationship of non-specific accumulation and the structural property of two type of 99m TC labeled compounds: (a) 99m Tc-(amine o xime) containing either 2-nitroimidazole (2-NI, as hypoxia tumor cells specific agents), or 4-nitro- imidazole (4-NI, as control), or aniline (as reference) groups; (b) 99m Tc-(arginine-glycine- aspartic acid, RGD, as tumor angiogenesis specific agents) and 99m Tc-(arginine-glycine- glutarmic acid, RGE, as control). The 99m Tc-(amine-oxime) complexes, in addition to the 2-NI, 4-NI, and aniline groups, contain methyl-, ethyl-, propyl-, iso-butyl-, t-butyl-, phenyl-, and Benzyl- groups as well to make the radiotracers differing in structure and in lipophilicity , while the lipophilicity of a radiotracer plays an important role in non-specific cellular accumulation and protein binding, The results demonstrated that (1) the complex containing 2-NI showed specific

  8. Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

    Science.gov (United States)

    Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

    2017-10-01

    Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. DMPD: Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available eptor for LPS/LBP complexes: a short review. Schumann RR. Res Immunol. 1992 Jan;143(1):11-5. (.png) (.svg) (...ride (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. Authors Schuma.../LBP complexes: a short review. PubmedID 1373512 Title Function of lipopolysaccha....html) (.csml) Show Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS

  10. Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

    Science.gov (United States)

    Cohen-Dvashi, Hadas; Israeli, Hadar; Shani, Orly; Katz, Aliza; Diskin, Ron

    2016-11-15

    To effectively infect cells, Lassa virus needs to switch in an endosomal compartment from its primary receptor, α-dystroglycan, to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have an impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity, it is not required for spike-mediated membrane fusion per se Our studies reveal important regulatory roles for histidines from the triad in sensing acidic pH and preventing premature spike triggering. We further show that LAMP1 requires a positively charged His230 residue to engage with the spike complex and that LAMP1 binding promotes membrane fusion. These results elucidate the molecular role of LAMP1 binding during Lassa virus cell entry and provide new insights into how pH is sensed by the spike. Lassa virus is a devastating disease-causing agent in West Africa, with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years, we learned that Lassa virus needs to switch receptors in a pH-dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effects of switching were known. Here we investigate the activity of the viral spike complex after abrogation of its ability to switch receptors. These studies inform us about the role of switching receptors and provide new insights into how the spike senses acidic pH. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  12. PRODIGY : a web server for predicting the binding affinity of protein-protein complexes

    NARCIS (Netherlands)

    Xue, Li; Garcia Lopes Maia Rodrigues, João; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-01-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given

  13. Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

    DEFF Research Database (Denmark)

    Madsen, Kenneth L; Beuming, Thijs; Niv, Masha Y

    2005-01-01

    PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (te...

  14. High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidicacid

    DEFF Research Database (Denmark)

    Khandelia, Himanshu

    2008-01-01

    , comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of XPA ) 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 ( 80 × 106. An MD simulation on the siramesine:PA interaction...

  15. NMR assignments of juvenile hormone binding protein in complex with JH III.

    Science.gov (United States)

    Suzuki, Rintaro; Tase, Akira; Fujimoto, Zui; Shiotsuki, Takahiro; Yamazaki, Toshimasa

    2009-06-01

    A hemolymph juvenile hormone binding protein (JHBP) shuttles hydrophobic JH, a key hormone in regulation of the insect life cycle, from the site of the JH biosynthesis to the cells of target organs. We report complete NMR chemical shift assignments of Bombyx mori JHBP in the JH III-bound state.

  16. Large-scale quantitative assessment of binding preferences in protein - nucleic acid complexes

    Czech Academy of Sciences Publication Activity Database

    Jakubec, Dávid; Vondrášek, Jiří

    2015-01-01

    Roč. 22, č. 1 (2015), s. 12 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 Keywords : DNA * binding preferences * ab initio calculations Subject RIV: CE - Biochemistry

  17. Effect of Binding Components in Complex Sample Matrices on Recovery in Direct Immersion Solid-Phase Microextraction: Friends or Foe?

    Science.gov (United States)

    Alam, Md Nazmul; Pawliszyn, Janusz

    2018-02-20

    The development of matrix compatible coatings for solid-phase microextraction (SPME) has enabled direct extraction of analytes from complex sample matrices. The direct immersion (DI) mode of SPME when utilized in conjunction with such extraction phases facilitates extraction of a wide range of analytes from complex matrices without the incurrence of fouling or coating saturation. In this work, mathematical models and computational simulations were employed to investigate the effect of binding components present in complex samples on the recovery of small molecules varying in logP for extractions carried out using the direct immersion approach. The presented findings corroborate that the studied approach indeed enables the extraction of both polar and nonpolar analytes from complex matrices, provided a suitable sorbent is employed. Further results indicated that, in certain cases, the kinetics of extraction of a given analyte in its free form might be dependent on the desorption kinetics of their bound form from matrix components, which might lower total recoveries of analytes with high affinity for the matrix. However, the binding of analytes to matrix components also enables SPME to extract a balanced quantity of different logP analytes, facilitated by multiphase equilibria, with a single extraction device.

  18. Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone

    Energy Technology Data Exchange (ETDEWEB)

    Mao, Yang; Xu, Xianzhong; Xu, Wei; Ishida, Yuko; Leal, Walter S.; Ames, James B.; Clardy, Jon (Harvard-Med); (UCD)

    2010-11-15

    Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the female's antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBP-mediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP {center_dot} pheromone complex. Although CquiOBP1 binds MOP in a pH-dependent fashion, it lacks the C terminus required for the pH-dependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.

  19. GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain.

    Science.gov (United States)

    Tornow, J; Zeng, X; Gao, W; Santangelo, G M

    1993-01-01

    In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1. Images PMID:8508768

  20. Binding abilities of a chiral calix[4]resorcinarene: a polarimetric investigation on a complex case of study

    Directory of Open Access Journals (Sweden)

    Marco Russo

    2017-12-01

    Full Text Available Polarimetry was used to investigate the binding abilities of a chiral calix[4]resorcinarene derivative, bearing L-proline subunits, towards a set of suitably selected organic guests. The simultaneous formation of 1:1 and 2:1 host–guest inclusion complexes was observed in several cases, depending on both the charge status of the host and the structure of the guest. Thus, the use of the polarimetric method was thoroughly revisited, in order to keep into account the occurrence of multiple equilibria. Our data indicate that the stability of the host–guest complexes is affected by an interplay between Coulomb interactions, π–π interactions, desolvation effects and entropy-unfavorable conformational dynamic restraints. Polarimetry is confirmed as a very useful and versatile tool for the investigation of supramolecular interactions with chiral hosts, even in complex systems involving multiple equilibria.

  1. New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

    Science.gov (United States)

    Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

    2004-11-21

    Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred).

  2. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    Science.gov (United States)

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  3. Rhenium complexes of chromophore-appended dipicolylamine ligands: syntheses, spectroscopic properties, DNA binding and X-ray crystal structure

    International Nuclear Information System (INIS)

    Mullice, L.A.; Buurma, N.J.; Pope, S.J.A.; Laye, R.H.; Harding, L.P.

    2008-01-01

    The syntheses of two chromophore-appended dipicolylamine-derived ligands and their reactivity with penta-carbonyl-chloro-rhenium have been studied. The resultant complexes each possess the fac-Re(CO) 3 core. The ligands L 1 1-[bis(pyridine-2-yl-methyl)amino]methyl-pyrene and L 2 2-[bis(pyridine-2-yl-methyl)amino]methyl-quinoxaline were isolated via a one-pot reductive amination in moderate yield. The corresponding rhenium complexes were isolated in good yields and characterised by 1 H NMR, MS, IR and UV-Vis studies. X-Ray crystallographic data were obtained for fac-{Re(CO) 3 (L 1 )}(BF 4 ), C 34 H 26 BF 4 N 4 O 3 Re: monoclinic, P2(1)/c, a 18.327(2) Angstroms, α = 90.00 degrees, b 14.1537(14) Angstroms, β96.263(6) degrees, c = 23.511(3) Angstroms, γ 90.00 Angstroms, 6062.4(11) (Angstroms) 3 , Z=8. The luminescence properties of the ligands and complexes were also investigated, with the emission attributed to the appended chromophore in each case. Isothermal titration calorimetry suggests that fac-{Re(CO) 3 (L 1 )}(BF 4 ) self-aggregates cooperatively in aqueous solution, probably forming micelle-like aggregates with a cmc of 0.18 mM. Investigations into the DNA-binding properties of fac-{Re(CO) 3 (L 1 )}(BF 4 ) were undertaken and revealed that fac-{Re(CO) 3 (L 1 )}(BF 4 ) binding to fish sperm DNA (binding constant 1.5 ± 0.2 * 10 5 M -1 , binding site size 3.2 ± 0.3 base pairs) is accompanied by changes in the UV-Vis spectrum as typically observed for pyrene-based intercalators while the calorimetrically determined binding enthalpy (-14 ± 2 kcal mol -1 ) also agrees favourably with values as typically found for intercalators. (authors)

  4. Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice.

    Directory of Open Access Journals (Sweden)

    Naïg Gueguen

    Full Text Available Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5μM, resveratrol stimulated complex I activity, whereas at high dose (50 μM it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed.

  5. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    International Nuclear Information System (INIS)

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni; Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin; Jiang, Tao

    2006-01-01

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents

  6. Spectroscopic methods for aqueous cyclodextrin inclusion complex binding measurement for 1,4-dioxane, chlorinated co-contaminants, and ozone

    Science.gov (United States)

    Khan, Naima A.; Johnson, Michael D.; Carroll, Kenneth C.

    2018-03-01

    Recalcitrant organic contaminants, such as 1,4-dioxane, typically require advanced oxidation process (AOP) oxidants, such as ozone (O3), for their complete mineralization during water treatment. Unfortunately, the use of AOPs can be limited by these oxidants' relatively high reactivities and short half-lives. These drawbacks can be minimized by partial encapsulation of the oxidants within a cyclodextrin cavity to form inclusion complexes. We determined the inclusion complexes of O3 and three common co-contaminants (trichloroethene, 1,1,1-trichloroethane, and 1,4-dioxane) as guest compounds within hydroxypropyl-β-cyclodextrin. Both direct (ultraviolet or UV) and competitive (fluorescence changes with 6-p-toluidine-2-naphthalenesulfonic acid as the probe) methods were used, which gave comparable results for the inclusion constants of these species. Impacts of changing pH and NaCl concentrations were also assessed. Binding constants increased with pH and with ionic strength, which was attributed to variations in guest compound solubility. The results illustrate the versatility of cyclodextrins for inclusion complexation with various types of compounds, binding measurement methods are applicable to a wide range of applications, and have implications for both extraction of contaminants and delivery of reagents for treatment of contaminants in wastewater or contaminated groundwater.

  7. Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

    NARCIS (Netherlands)

    Wilczak, N; Kuhl, N; Chesik, D; Geerts, A; Luiten, P; De Keyser, J

    The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R-3 -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were

  8. Tuning affinity and reversibility for O2 binding in dinuclear Co(II) complexes

    DEFF Research Database (Denmark)

    Vad, Mads Sørensen; Johansson, Frank Bartnik; Seidler-Egdal, Rune Kirk

    2013-01-01

    The O2 binding affinity of a series of dicobalt(II) complexes can be tuned between p(O2)50% = 2.3 × 10−3 and 700 × 10−3 atm at 40 °C by varying the number of H and Cl atoms in the bridging acetato ligands of [Co2(bpbp)(CH(3−n)ClnCO2)(CH3CN)2]2+, where bpbp− = 2,6-bis(N,N-bis(2-pyridylmethyl)amino...

  9. Structure-function relations in oxaloacetate decarboxylase complex. Fluorescence and infrared approaches to monitor oxomalonate and Na(+ binding effect.

    Directory of Open Access Journals (Sweden)

    Thierry Granjon

    Full Text Available BACKGROUND: Oxaloacetate decarboxylase (OAD is a member of the Na(+ transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na(+ ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of alpha, beta and gamma subunits. The alpha subunit contains the carboxyltransferase catalytic site. METHODOLOGY/PRINCIPAL FINDINGS: In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate and Na(+ effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES, indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na(+. REES changes due to oxomalonate binding were also observed with the alphagamma and alpha subunits. Infrared spectra showed that OAD, alphagamma and alpha subunits have a main component band centered between 1655 and 1650 cm(-1 characteristic of a high content of alpha helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of beta sheet structures and a concomitant increase of alpha helix structures. Oxomalonate binding to alphagamma and alpha subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. CONCLUSION: Oxomalonate binding affects the

  10. The Panitumumab EGFR Complex Reveals a Binding Mechanism That Overcomes Cetuximab Induced Resistance.

    Directory of Open Access Journals (Sweden)

    E Allen Sickmier

    Full Text Available Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation.

  11. Synthesis, CMC Determination, Antimicrobial Activity and Nucleic Acid Binding of A Surfactant Copper(II) Complex Containing Phenanthroline and Alanine Schiff-Base.

    Science.gov (United States)

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-03-01

    A new water-soluble surfactant copper(II) complex [Cu(sal-ala)(phen)(DA)] (sal-ala = salicylalanine, phen = 1,10-phenanthroline, DA = dodecylamine), has been synthesized and characterized by physico-chemical and spectroscopic methods. The critical micelle concentration (CMC) values of this surfactant-copper(II) complex in aqueous solution were obtained from conductance measurements. Specific conductivity data (at 303, 308, 313. 318 and 323 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔG(0)m, ΔH(0)m and ΔS(0)m). The interaction of this complex with nucleic acids (DNA and RNA) has been explored by using electronic absorption spectral titration, competitive binding experiment, cyclic voltammetry, circular dichroism (CD) spectra, and viscosity measurements. Electronic absorption studies have revealed that the complex can bind to nucleic acids by the intercalative binding mode which has been verified by viscosity measurements. The DNA binding constants have also been calculated (Kb = 1.2 × 10(5) M(-1) for DNA and Kb = 1.6 × 10(5) M(-1) for RNA). Competitive binding study with ethidium bromide (EB) showed that the complex exhibits the ability to displace the DNA-bound-EB indicating that the complex binds to DNA in strong competition with EB for the intercalative binding site. The presence of hydrophobic ligands, alanine Schiff-base, phenanthroline and long aliphatic chain amine in the complex were responsible for this strong intercalative binding. The surfactant-copper (II) complex was screened for its antibacterial and antifungal activities against various microorganisms. The results were compared with the standard drugs, amikacin(antibacterial) and ketokonazole(antifungal).

  12. Genome-wide identification and characterization of Notch transcription complex-binding sequence paired sites in leukemia cells

    Science.gov (United States)

    Severson, Eric; Arnett, Kelly L.; Wang, Hongfang; Zang, Chongzhi; Taing, Len; Liu, Hudan; Pear, Warren S.; Liu, X. Shirley; Blacklow, Stephen C.; Aster, Jon C.

    2018-01-01

    Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and are linked to the Notch-responsiveness of a few genes, but their overall contribution to Notch-dependent gene regulation is unknown. To address this issue, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay, and applied insights from these in vitro studies to Notch-“addicted” leukemia cells. We find that SPSs contribute to the regulation of approximately a third of direct Notch target genes. While originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5. Our work provides a general method for identifying sequence-paired sites in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells. PMID:28465412

  13. Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex

    Directory of Open Access Journals (Sweden)

    Walaa Oweis

    2016-09-01

    Full Text Available Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s. Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.

  14. RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

    Science.gov (United States)

    Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

    2013-04-16

    The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

  15. Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.

    Science.gov (United States)

    Stroud, Amy; Liddell, Susan; Allers, Thorsten

    2012-01-01

    Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a five-stranded β-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as replication protein A (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3) exist in operons with a novel gene specific to Euryarchaeota; this gene encodes a protein that we have termed RPA-associated protein (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only Δrpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs). We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

  16. Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

    Science.gov (United States)

    Moghanibashi, Mehdi; Rastgar Jazii, Ferdous; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

    2013-06-01

    Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

  17. Hierarchy and Assortativity as New Tools for Binding-Affinity Investigation: The Case of the TBA Aptamer-Ligand Complex.

    Science.gov (United States)

    Cataldo, Rosella; Alfinito, Eleonora; Reggiani, Lino

    2017-12-01

    Aptamers are single stranded DNA, RNA, or peptide sequences having the ability to bind several specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually, they are generated in vitro, although computational approaches have been recently developed for the in silico production. Despite these efforts, the mechanism of aptamer-ligand formation is not completely clear, and producing high-affinity aptamers is still quite difficult. This paper aims to develop a computational model able to describe aptamer-ligand affinity. Topological tools, such as the conventional degree distribution, the rank-degree distribution (hierarchy), and the node assortativity are employed. In doing so, the macromolecules tertiary-structures are mapped into appropriate graphs. These graphs reproduce the main topological features of the macromolecules, by preserving the distances between amino acids (nucleotides). Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex produced in the presence of Na + or K + . The topological analysis is able to detect several differences between complexes obtained in the presence of the two cations, as expected by previous investigations. These results support graph analysis as a novel computational tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, revealing that the resistance is sensitively affected by the presence of sodium or potassium, thus suggesting resistance as a useful physical parameter for testing binding affinity.

  18. Inclusion complexes of azadirachtin with native and methylated cyclodextrins: solubilization and binding ability.

    Science.gov (United States)

    Liu, Yu; Chen, Guo-Song; Chen, Yong; Lin, Jun

    2005-06-02

    The inclusion complexation behavior of azadirachtin with several cyclodextrins and their methylated derivatives has been investigated in both solution and the solid state by means of XRD, TG-DTA, DSC, NMR, and UV-vis spectroscopy. The results show that the water solubility of azadirachtin was obviously increased after resulting inclusion complex with cyclodextrins. Typically, beta-cyclodextrin (beta-CD), dimethyl-beta-cyclodextrin (DMbetaCD), permethyl-beta-cyclodextrin (TMbetaCD), and hydroxypropyl-beta-cyclodextrin (HPbetaCD) are found to be able to solubilize azadirachtin to high levels up to 2.7, 1.3, 3.5, and 1.6 mg/mL (calculated as azadirachtin), respectively. This satisfactory water solubility and high thermal stability of the cyclodextrin-azadirachtin complexes, will be potentially useful for their application as herbal medicine or healthcare products.

  19. Mutations at the Qo-Site of the Cytochrome bc1 Complex Strongly Affect Oxygen Binding

    DEFF Research Database (Denmark)

    Husen, Peter; Solov'yov, Ilia A

    2017-01-01

    The homodimeric bc1 protein complex is embedded in membranes of mitochondria and photosynthetic bacteria, where it transports protons across the membrane to maintain an electrostatic potential used to drive ATP synthesis as part of the respiratory or photosynthetic pathways. The reaction cycle...... at the Qo-sites, and, moreover, different behavior of the two monomers of the bc1 complex is observed. The conformational differences at the Qo-sites of the two monomers are studied in detail and discussed. The anionic form of semiquinone was identified as leading to the greatest opportunity for side...

  20. Simplifying complex sequence information: a PCP-consensus protein binds antibodies against all four Dengue serotypes.

    Science.gov (United States)

    Bowen, David M; Lewis, Jessica A; Lu, Wenzhe; Schein, Catherine H

    2012-09-14

    Designing proteins that reflect the natural variability of a pathogen is essential for developing novel vaccines and drugs. Flaviviruses, including Dengue (DENV) and West Nile (WNV), evolve rapidly and can "escape" neutralizing monoclonal antibodies by mutation. Designing antigens that represent many distinct strains is important for DENV, where infection with a strain from one of the four serotypes may lead to severe hemorrhagic disease on subsequent infection with a strain from another serotype. Here, a DENV physicochemical property (PCP)-consensus sequence was derived from 671 unique sequences from the Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein (EdomIII) were expressed from synthetic genes in Escherichia coli. The ability of the purified consensus proteins to bind polyclonal antibodies generated in response to infection with strains from each of the four DENV serotypes was determined. The initial consensus protein bound antibodies from DENV-1-3 in ELISA and Western blot assays. This sequence was altered in 3 steps to incorporate regions of maximum variability, identified as significant changes in the PCPs, characteristic of DENV-4 strains. The final protein was recognized by antibodies against all four serotypes. Two amino acids essential for efficient binding to all DENV antibodies are part of a discontinuous epitope previously defined for a neutralizing monoclonal antibody. The PCP-consensus method can significantly reduce the number of experiments required to define a multivalent antigen, which is particularly important when dealing with pathogens that must be tested at higher biosafety levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Newsilver complexes with bioactive glycine and nicotinamide molecules – characterization, DNA binding, antimicrobial and anticancer evaluation

    Czech Academy of Sciences Publication Activity Database

    Rendošová, M.; Vargová, Z.; Kuchár, J.; Sabolová, D.; Levoča, Š.; Kudláčová, J.; Paulíková, H.; Hudecová, D.; Helebrandtová, V.; Almáši, M.; Vilková, M.; Dušek, Michal; Bobáľová, D.

    2017-01-01

    Roč. 168, Mar (2017), s. 1-12 ISSN 0162-0134 Institutional support: RVO:68378271 Keywords : silver complex * antimicrobial effect * stability * cytotoxicity * intercalation * topo I and II inhibition assay Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 3.348, year: 2016

  2. Pigment binding sites occupancy and functional architecture of the Photosystem II antenna complex Lhcb5

    NARCIS (Netherlands)

    Ballottari, M.; Mozzo, M.; Croce, R.; Morosinotto, T.; Bassi, R.

    2009-01-01

    Lhcb5 is an antenna protein that is highly conserved in plants and green algae. It is part of the inner layer of photosystem II antenna system retained in high light acclimated plants. To study the structure-function relation and the role of individual pigments in this complex, we (i) "knocked out"

  3. Circular Dichroism is Sensitive to Monovalent Cation Binding in Monensin Complexes

    Czech Academy of Sciences Publication Activity Database

    Nedzhib, A.; Kessler, Jiří; Bouř, Petr; Gyurcsik, B.; Pantcheva, I.

    2016-01-01

    Roč. 28, č. 5 (2016), s. 420-428 ISSN 0899-0042 R&D Projects: GA ČR GA15-09072S; GA ČR(CZ) GA16-05935S Institutional support: RVO:61388963 Keywords : monovalent polyether ionophore * metal complexes * synchrotron radiation circular dichroism * time-dependent density functional theory Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.956, year: 2016

  4. Structural aspects of inotropic bipyridine binding. Crystal structure determination to 1.9 A of the human serum transthyretin-milrinone complex.

    Science.gov (United States)

    Wojtczak, A; Luft, J R; Cody, V

    1993-03-25

    The crystal structure of human transthyretin (TTR) complexed with milrinone (2-methyl-5-cyano-3,4'-bipyridin-6(1H)-one), a positive inotropic cardiac agent, has been refined to R = 17.4% for 8-1.9-A resolution data. This report provides the first detailed description of protein interactions for an inotropic bipyridine agent which is an effective thyroid hormone binding competitor to transthyretin. Milrinone is bound along the 2-fold axis in the binding site with its substituted pyridone ring located deep within the channel of the two identical binding domains of the TTR tetramer. In this orientation the 5-cyano group occupies the same site as the 3'-iodine in the TTR complex with 3,3'-diiodothyronine (Wojtczak, A., Luft, J., and Cody, V. (1992) J. Biol. Chem. 267, 353-357), which is 3.5 A deeper in the channel than thyroxine (Blake, C. C. F., and Oately, S. J., (1977) Nature 268, 115-120). These structural results confirm computer modeling studies of milrinone structural homology with thyroxine and its TTR binding interactions and explain the effectiveness of milrinone competition for thyroxine binding to TTR. To understand the weaker binding affinity of the parent inotropic drug, amrinone (5-amino-3,4'-bipyridin-6(1H)-one), modeling studies of its TTR binding were carried out which indicate that the 5-amino group cannot participate in strong interactions with TTR and the lack of the 2-methyl further weakens amrinone binding.

  5. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

    Energy Technology Data Exchange (ETDEWEB)

    Hirose, Mika; Sugiyama, Shigeru, E-mail: sugiyama@chem.eng.osaka-u.ac.jp [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Ishida, Hanako; Niiyama, Mayumi [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Daisuke; Hara, Toshiaki [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Mizohata, Eiichi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Murakami, Satoshi [Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagaw 226-8501 (Japan); Inoue, Tsuyoshi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Shigeru; Murata, Michio [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan)

    2013-11-01

    The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

  6. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

    International Nuclear Information System (INIS)

    Hirose, Mika; Sugiyama, Shigeru; Ishida, Hanako; Niiyama, Mayumi; Matsuoka, Daisuke; Hara, Toshiaki; Mizohata, Eiichi; Murakami, Satoshi; Inoue, Tsuyoshi; Matsuoka, Shigeru; Murata, Michio

    2013-01-01

    The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS

  7. Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5.

    Science.gov (United States)

    Sun, Min; Long, Juan; Yi, Yuxin; Xia, Wei

    2017-10-28

    Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-β: K D =2.44e-7, IGFBP-5/importin-α5: K D =3.4e-7). Blocking the importin-α5/importin-β nuclear import pathway using SiRNA or dominant negative impotin-β dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5 ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5 ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-β complex, and NLS is critical domain in IGFBP-5 nuclear translocation.

  8. Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

    Science.gov (United States)

    Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

    2014-03-07

    The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

  9. APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane.

    Science.gov (United States)

    Mills, Kate M; Brocardo, Mariana G; Henderson, Beric R

    2016-02-01

    Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. © 2016 Mills et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Mistarz, Ulrik H; Brown, Jeffery M; Haselmann, Kim F

    2016-01-01

    Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-milliseco......Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub......-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution...

  11. Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex.

    Science.gov (United States)

    Aguero, Tristan; Jin, Zhigang; Chorghade, Sandip; Kalsotra, Auinash; King, Mary Lou; Yang, Jing

    2017-10-15

    In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development. © 2017. Published by The Company of Biologists Ltd.

  12. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    . The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  13. Cooperativity and binding isotherms of the amylose-iodine-triiodide complex

    International Nuclear Information System (INIS)

    Cesaro, A.; Benegas, J.C.; Ripoll, D.R.

    1984-08-01

    A simple model has been presented which is based on some widely accepted characteristics of the amylose-iodine-triiodide complex and is capable of correlating a number of experimental features with molecular parameters. A substantial improvement is obtained removing weakness and arbitrariness of some adjustable parameters of previous models. The proposed model assumes that sequences of bound species are initiated by triiodide ions and propagated (principally) by iodine. The theory is developed by using the matrix method of Zimm and Bragg and assuming that the cooperative interaction is of electrostatic nature. The model reproduces most of the experimental data and predicts a finite length of the bound polyiodine chain for any length of the host polymer. (author)

  14. The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

    Science.gov (United States)

    Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

    2000-08-01

    The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

  15. Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission

    OpenAIRE

    Kittler, Josef T.; Chen, Guojun; Honing, Stephan; Bogdanov, Yury; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Jovanovic, Jasmina N.; Pangalos, Menelas N.; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2005-01-01

    The efficacy of synaptic inhibition depends on the number of γ-aminobutyric acid type A receptors (GABAARs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABAAR endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABAAR β subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the β3 subunit) incorporates...

  16. Multispectroscopic DNA-Binding studies and antimicrobial evaluation of new mixed-ligand Silver(I) complex and nanocomplex: A comparative study

    Science.gov (United States)

    Movahedi, Elaheh; Rezvani, Ali Reza

    2018-05-01

    A novel mixed-ligand Ag(I) complex, , has been synthesized and characterized by the elemental analysis, IR spectroscopy and 1HNMR. In the formula, dian and phen are N-(4,5-diazafluoren-9-ylidene)aniline and 1,10-phenanthroline, respectively. This complex also has been prepared at nano size by sonochemical technique and characterized by the FTIR and scanning electron microscopy (SEM). To evaluate the biological preferences of the Ag(I) complex and nanocomplex and verify the relationships between the structure and biological function, in vitro DNA binding and antibacterial experiments have been carried out. DNA-complex interaction has been pursued by electronic absorption titration, luminescence titration, competitive binding experiment, effect of ionic strength, thermodynamic studies, viscometric evaluation and circular dichroism spectroscopy in the physiological pH. Each compound displays significant binding trend to the CT-DNA. The mode of binding to the CT-DNA probably is a moderate intercalation mode with the partial insertion of the planar ligands between the base stacks of double-stranded DNA. The relative viscosities and circular dichroism spectra of the CT-DNA with the complex solutions, confirm the intense interactions of the Ag(I) complex and nanocomplex with DNA. An in vitro antibacterial test of the complex and nanocomplex on a series of the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) shows a remarkable antibacterial feature of the Ag(I) complex. The MIC values (minimum inhibitory concentration) of the compounds compare with silver nitrate and silver sulfadiazine. The bacterial inhibitions of the Ag(I) complex and nanocomplex are agreed to their DNA binding affinities.

  17. Circulating immune complexes in the serum in systemic lupus erythematosus and in carriers of hepatitis B antigen: quantitation by binding to radiolabeled Cl/sub q/

    International Nuclear Information System (INIS)

    Nydegger, U.E.; Lambert, P.H.; Gerber, H.; Miescher, P.A.

    1974-01-01

    A sensitive and reproducible procedure fr the detection of souble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled Clq is reacted with sera containing immune complexes. Separation of free from complex bound [ 125 I]Clq is achieved by selective precipitation with polyethylene glycol (PEG). The minimal amount of aggregated immunoglobulins thus detected is about 10 μg and that of soluble human IgG-anti-IgG complexes is about 3 μg of complexed antibody. Some immune complexes formed in large antigen excess (Ag 2 Ab) can still be detected by this radiolabeled Clq bining assay. In a second step, this radiolabeled Clq binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes. Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the Hepatitis B antigen (HB-Ag). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients. No increased [ 125 I]Clq binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [ 125 I]Clq percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [ 125 I]Clq. The results were also used for a correlative study of [ 125 I]Clq binding to IgG levels in the sera but increased [ 125 I]Clq binding could not be attributed to high serum IgG levels which are likely to account for the evaluation of immune complex diseases in human pathology. (U.S.)

  18. Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

    Science.gov (United States)

    Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

    2009-01-19

    A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

  19. Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine.

    Science.gov (United States)

    Esteghamat-Panah, Roya; Hadadzadeh, Hassan; Farrokhpour, Hossein; Simpson, Jim; Abdolmaleki, Amir; Abyar, Fatemeh

    2017-02-15

    A new mononuclear rhodium(III) complex, [Rh(bzimpy)Cl 3 ] (bzimpy = 2,6-bis(2-benzimidazolyl)pyridine), was synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with fish sperm DNA (FS-DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement in order to evaluate the possible DNA-binding mode and to calculate the corresponding DNA-binding constant. The results reveal that the Rh(III) complex interacts with DNA through groove binding mode with a binding affinity on the order of 10 4 . In addition, the binding of the Rh(III) complex to bovine serum albumin (BSA) was monitored by UV-Vis and fluorescence emission spectroscopy at different temperatures. The mechanism of the complex interaction was found to be static quenching. The thermodynamic parameters (ΔH, ΔS, and ΔG) obtained from the fluorescence spectroscopy data show that van der Waals interactions and hydrogen bonds play a major role in the binding of the Rh(III) complex to BSA. For the comparison of the DNA- and BSA-binding affinities of the free bzimpy ligand with its Rh(III) complex, the absorbance titration and fluorescence quenching experiments of the free bzimpy ligand with DNA and BSA were carried out. Competitive experiments using eosin Y and ibuprofen as site markers indicated that the complex was mainly located in the hydrophobic cavity of site I of the protein. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the Rh(III) complex against the MCF-7, K562, and HT-29 cell lines were evaluated and compared with those of the free ligand (bzimpy). It was found that the complexation process improved the anticancer activity significantly. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: Residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family

    International Nuclear Information System (INIS)

    Contessa, Gian Marco; Orsale, Maria; Melino, Sonia; Torre, Vincent; Paci, Maurizio; Desideri, Alessandro; Cicero, Daniel O.

    2005-01-01

    The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs

  1. 'In-Crystallo' Capture of a Michaelis Complex And Product Binding Modes of a Bacterial Phosphotriesterase

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, C.J.; Foo, J.-L.; Kim, H.-K.; Carr, P.D.; Liu, J.-W.; Salem, G.; Ollis, D.L.

    2009-05-18

    The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The {mu}-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that {mu}-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favor attack from a solvent molecule terminally coordinated to the {alpha}-metal ion. The bridging of both metal ions by the product, without disruption of the {mu}-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the {beta}-metal ion, displacing the {mu}-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a {mu}-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK{sub a} for the nucleophile, consistent with a general base function during catalysis.

  2. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    Science.gov (United States)

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  3. Control of flowering and cell fate by LIF2, an RNA binding partner of the polycomb complex component LHP1.

    Directory of Open Access Journals (Sweden)

    David Latrasse

    Full Text Available Polycomb Repressive Complexes (PRC modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein like heterochromatin protein1 (LHP1 is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2. LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA

  4. Flexible Acyclic Polyol-Chloride Anion Complexes and Their Characterization by Photoelectron Spectroscopy and Variable Temperature Binding Constant Determinations

    Energy Technology Data Exchange (ETDEWEB)

    Shokri, Alireza; Wang, Xue B.; Wang, Yangping; O' Doherty, George A.; Kass, Steven R.

    2016-03-17

    Flexible acyclic alcohols with 1–5 hydroxyl groups were bound to chloride anion and these complexes were interrogated by negative ion photoelectron spectroscopy and companion density functional theory computations. The resulting vertical detachment energies are reproduced on average to 0.10 eV by M06-2X/aug-cc-pVTZ predictions and range from 4.45 – 5.96 eV. These values are 0.84 – 2.35 eV larger than the adiabatic detachment energy of Cl– as a result of the larger hydrogen bond networks in the bigger polyols. Adiabatic detachment energies of the alcohol–Cl– clusters are more difficult to determine both experimentally and computationally. This is due to the large geometry changes that occur upon photodetachment and the large bond dissociation energy of H–Cl which enables the resulting chlorine atom to abstract a hydrogen from any of the methylene (CH2) or methine (CH) positions. Both ionic and non-ionic hydrogen bonds (i.e., OH•••Cl– and OH•••OH•••Cl–) form in the larger polyols complexes, and are found to be energetically comparable. Subtle structural differences, consequently can lead to the formation of different types of hydrogen bonds and maximizing the ionic ones is not always preferred. Solution equilibrium binding constants between the alcohols and tetrrabuylammonium chloride (TBACl) in acetonitrile at -24.2, 22.0, and 53.6 °C were also determined. The free energies of association are nearly identical for all of the substrates (i.e., ΔG° = -2.8 ± 0.7 kcal mol–1). Compensating enthalpy and entropy values reveal, contrary to expectation and the intrinsic gas-phase preferences, that the bigger systems with more hydroxyl groups are entropically favored and enthalpically disfavored relative to the smaller species. This suggests that more solvent molecules are released upon binding TBACl to alcohols with more hydroxyl groups and is consistent with the measured negative heat capacities. These quantities increase with

  5. Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens.

    Science.gov (United States)

    Wikman, Maria; Friedman, Mikaela; Pinitkiatisakul, Sunan; Hemphill, Andrew; Lövgren-Bengtsson, Karin; Lundén, Anna; Ståhl, Stefan

    2005-04-01

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native

  6. DNA and protein binding, double-strand DNA cleavage and cytotoxicity of mixed ligand copper(II) complexes of the antibacterial drug nalidixic acid.

    Science.gov (United States)

    Loganathan, Rangasamy; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Muruganantham, Amsaveni; Ghosh, Swapan K; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkader

    2017-09-01

    The water soluble mixed ligand complexes [Cu(nal)(diimine)(H 2 O)](ClO 4 ) 1-4, where H(nal) is nalidixic acid and diimine is 2,2'-bipyridine (1), 1,10-phenanthroline (2), 5,6-dimethyl-1,10-phenanthroline (3), and 3,4,7,8-tetramethyl-1,10-phenanthroline (4), have been isolated. The coordination geometry around Cu(II) in 1 and that in the Density Functional Theory optimized structures of 1-4 has been assessed as square pyramidal. The trend in DNA binding constants (K b ) determined using absorption spectral titration (K b : 1, 0.79±0.1base pair. In contrast, 3 and 4 are involved in intimate hydrophobic interaction with DNA through the methyl substituents on phen ring, which is supported by viscosity and protein binding studies. DNA docking studies imply that 4 is involved preferentially in DNA major groove binding while 1-3 in minor groove binding and that all the complexes, upon removing the axially coordinated water molecule, bind in the major groove. Interestingly, 3 and 4 display prominent double-strand DNA cleavage while 1 and 2 effect only single-strand DNA cleavage in the absence of an activator. The complexes 3 and 4 show cytotoxicity higher than 1 and 2 against human breast cancer cell lines (MCF-7). The complex 4 induces apoptotic mode of cell death in cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Synthesis and Structural Investigation of New Bio-Relevant Complexes of Lanthanides with 5-Hydroxyflavone: DNA Binding and Protein Interaction Studies

    Directory of Open Access Journals (Sweden)

    Alexandra-Cristina Munteanu

    2016-12-01

    Full Text Available In the present work, we attempted to develop new metal coordination complexes of the natural flavonoid 5-hydroxyflavone with Sm(III, Eu(III, Gd(III, Tb(III. The resultant hydroxo complexes have been characterized by a variety of spectroscopic techniques, including fluorescence, FT-IR, UV-Vis, EPR and mass spectral studies. The general chemical formula of the complexes is [Ln(C15H9O33(OH2(H2Ox]·nH2O, where Ln is the lanthanide cation and x = 0 for Sm(III, x = 1 for Eu(III, Gd(III, Tb(III and n = 0 for Sm(III, Gd(III, Tb(III, n = 1 for Eu(III, respectively. The proposed structures of the complexes were optimized by DFT calculations. Theoretical calculations and experimental determinations sustain the proposed structures of the hydroxo complexes, with two molecules of 5-hydroxyflavone acting as monoanionic bidentate chelate ligands. The interaction of the complexes with calf thymus DNA has been explored by fluorescence titration and UV-Vis absorption binding studies, and revealed that the synthesized complexes interact with DNA with binding constants (Kb ~ 104. Human serum albumin (HSA and transferrin (Tf binding studies have also been performed by fluorescence titration techniques (fluorescence quenching studies, synchronous fluorescence spectra. The apparent association constants (Ka and thermodynamic parameters have been calculated from the fluorescence quenching experiment at 299 K, 308 K, and 318 K. The quenching curves indicate that the complexes bind to HSA with smaller affinity than the ligand, but to Tf with higher binding affinities than the ligand.

  8. Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

    Science.gov (United States)

    Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

    2006-11-24

    The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

  9. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

    Science.gov (United States)

    Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

    2016-01-01

    The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

  10. Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

    Science.gov (United States)

    Wieczorek, Anna; Downey, Christopher D; Dallmann, H Garry; McHenry, Charles S

    2010-09-17

    The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

  11. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  12. Extracellular fibrinogen-binding protein (Efb) from staphylococcus aureus Inhibits the formation of platelet-leukocyte complexes

    NARCIS (Netherlands)

    Posner, M.G; Upadhyay, A.; Abubaker, A.A.; Fortunato, T.M.; Vara, D.; Canobbio, I.; Bagby, S.; Pula, G.

    2016-01-01

    Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to

  13. Boronic acid adducts of technetium dioxime (BATO) complexes derived from quinuclidine benzilate (QNB) boronic acid stereoisomers: Syntheses and studies of their binding to the muscarinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Jurisson, Silvia S.; Pirro, John; DiRocco, Richard J.; Rosenspire, Karen C.; Jagoda, Elaine; Nanjappan, Palaniappa; Eckelman, William C.; Nowotnik, David P.; Nunn, Adrian D.

    1995-01-01

    We have investigated the possibility of using BATO complexes derivatized with the muscarinic acetylcholine receptor (mAChR) antagonist, quinuclidinyl benzilate (QNB), for mAChR imaging. The BATO complexes, TcCl(DMG) 3 B-QNB, were prepared using QNB derivatives containing a 4'-boronic acid substituent on one of the benzilic benzene rings (QNB-boronic acid). The QNB-boronic acid molecule has two chiral centers, and all four QNB-BATO stereoisomers were made and evaluated. When studied using in vitro receptor binding assays based on tissue from rat brain caudate-putamen (which contains primarily M 1 and M 4 mAChR) and rat heart (M 2 mAChR), the QNB-boronic acid stereoisomers had binding affinities (K A ) in the range 2 x 10 5 -1 x 10 8 , at least 10-fold lower than theK A for QNB (ca 2 x 10 9 ). The stereochemistry of both centers had some influence on the affinity constant. When the TcCl(DMG) 3 B-QNB complexes were studied, none of the stereoisomeric complexes displayed measurable specific binding (K A 6 ), but all showed high non-specific binding. In vitro autoradiography with rat brain slices confirmed the absence of specific binding in these tracers. In vivo, the 99m TcCl(DMG) 3 B-QNB complexes displayed minimal brain uptake, and modest heart uptake; the latter was unlikely to be related to uptake by the mAChR. In light of these findings, we conclude that the interaction between the TcCl(DMG) 3 B-QNB complexes and biological membranes is dominated by the hydrophobicity of the BATO moiety. The TcCl(DMG) 3 B-QNB complexes, therefore, have little potential for mAChR imaging

  14. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  15. Mononuclear zinc(II) complexes of 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols: Synthesis, structural characterization, DNA binding and cheminuclease activities

    Science.gov (United States)

    Ravichandran, J.; Gurumoorthy, P.; Karthick, C.; Kalilur Rahiman, A.

    2014-03-01

    Four new zinc(II) complexes [Zn(HL1-4)Cl2] (1-4), where HL1-4 = 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols, have been isolated and fully characterized using various spectro-analytical techniques. The X-ray crystal structure of complex 4 shows the distorted trigonal-bipyramidal coordination geometry around zinc(II) ion. The crystal packing is stabilized by intermolecular NH⋯O hydrogen bonding interaction. The complexes display no d-d electronic band in the visible region due to d10 electronic configuration of zinc(II) ion. The electrochemical properties of the synthesized ligands and their complexes exhibit similar voltammogram at reduction potential due to electrochemically innocent Zn(II) ion, which evidenced that the electron transfer is due to the nature of the ligand. Binding interaction of complexes with calf thymus DNA was studied by UV-Vis absorption titration, viscometric titration and cyclic voltammetry. All complexes bind with CT DNA by intercalation, giving the binding affinity in the order of 2 > 1 ≫ 3 > 4. The prominent cheminuclease activity of complexes on plasmid DNA (pBR322 DNA) was observed in the absence and presence of H2O2. Oxidative pathway reveals that the underlying mechanism involves hydroxyl radical.

  16. DNA binding and biological activity of mixed ligand complexes of Cu(II, Ni(II and Co(II with quinolones and N donor ligand

    Directory of Open Access Journals (Sweden)

    S.M M Akram

    2015-10-01

    Full Text Available  AbstractMixed ligand complexes of  Cu(II, Ni(II and Co(II have been synthesized by using levofloxacin and bipyridyl and characterized using spectral and analytical techniques. The binding behavior of the Ni(II and Cu(II complexes with herring sperm DNA(Hs-DNA were determined using electronic absorption titration, viscometric measurements and cyclic voltammetry measurements. The binding constant calculated  for Cu(II and Ni(II complexes are 2.0 x 104 and 4.0 x 104 M-1 respectively. Detailed analysis reveals that these metal complexes interact with DNA through intercalative binding mode. The nuclease activity of  Cu(II and Ni(II complexes with ct-DNA was carried out using agarose gel electrophoresis technique. The antioxidant activities for the synthesized complexes have been tested and the antibacterial activity for Ni(II complex was also checked.Key words: Intercalation, hypochromism, red shift and  peak potential.

  17. From simple receptors to complex multimodal percepts: a first global picture on the mechanisms involved in perceptual binding.

    Science.gov (United States)

    Velik, Rosemarie

    2012-01-01

    The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1) combination coding, (2) binding by synchrony, (3) population coding, (4) binding by attention, (5) binding by knowledge, expectation, and memory, (6) hardwired vs. on-demand binding, (7) bundling and binding of features, (8) the feature-integration theory of attention, and (9) synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1) at different hierarchical levels and (2) in different stages of "perceptual knowledge acquisition." The model furthermore considers and explains a number of inhibitory "filter mechanisms" that suppress the activation of inappropriate or currently irrelevant information.

  18. From simple receptors to complex multimodal percepts: A first global picture on the mechanisms involved in perceptual binding

    Directory of Open Access Journals (Sweden)

    Rosemarie eVelik

    2012-07-01

    Full Text Available The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1 combination coding, (2 binding by synchrony, (3 population coding, (4 binding by attention, (5 binding by knowledge, expectation, and memory, (6 hardwired versus on-demand binding, (7 bundling and binding of features, (8 the feature-integration theory of attention, (9 synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1 at different hierarchical levels and (2 in different stages of perceptual knowledge acquisition. The model furthermore considers and explains a number of inhibitory filter mechanisms that suppress the activation of inappropriate or currently irrelevant information.

  19. Low capacity of erythrocytes to bind with immune complexes via C3b receptor in patients with systemic lupus erythematosus: correlation with pathological proteinuria

    International Nuclear Information System (INIS)

    Nojima, Y.; Terai, C.; Minota, S.; Takano, K.; Miyakawa, Y.; Takaku, F.

    1985-01-01

    Erythrocytes from 51 patients with systemic lupus erythematosus and 75 controls were tested for the capacity to bind aggregated human gamma-globulin labeled with radioiodine in the presence of complement. Both in patients and controls, a trimodal distribution of binding capacity was observed. Low (less than 9% of the added radioactivity), intermediate (9-17%), and high binding (more than 17%) were observed in 13, 58, and 29% in controls and in 49, 43 and 8% in lupus patients. The low binding capacity of erythrocytes persisted even after patients entered remission following steroid therapy. A genetic control of binding capacity was supported by familial surveys. Prevalence of pathological proteinuria was significantly higher in patients with low binding capacity than those with intermediate or high binding capacity (16/25 vs 7/26, P less than 0.01). These results indicate that an impaired physiological disposal of immune complexes via the erythrocyte C3b receptor in lupus patients may contribute to the development of renal involvement

  20. Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface.

    Science.gov (United States)

    Allain, F; Denys, A; Spik, G

    1996-07-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.

  1. Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

    Full Text Available Abstract Background Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available. Results The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield, a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage λ genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence. Conclusion This study supports the conclusion that physics-based methods can offer a completely complementary

  2. Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

    Science.gov (United States)

    Patra, Ayan; Bera, Manindranath

    2014-01-30

    In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. DNA incision evaluation, binding investigation and biocidal screening of Cu(II), Ni(II) and Co(II) complexes with isoxazole Schiff bases.

    Science.gov (United States)

    Ganji, Nirmala; Chityala, Vijay Kumar; Marri, Pradeep Kumar; Aveli, Rambabu; Narendrula, Vamsikrishna; Daravath, Sreenu; Shivaraj

    2017-10-01

    Two new series of binary metal complexes [M(L 1 ) 2 ] and [M(L 2 ) 2 ] where, M=Cu(II), Ni(II) & Co(II) and L 1 =4-((3,4-dimethylisoxazol-5-ylimino)methyl)benzene-1,3-diol; L 2 =2-((3,4-dimethylisoxazol-5-ylimino)methyl)-5-methoxyphenol were synthesized and characterized by elemental analysis, 1 H NMR, 13 C NMR, FT-IR, ESI mass, UV-Visible, magnetic moment, ESR, SEM and powder XRD studies. Based on these results, a square planar geometry is assigned for all the metal complexes where the Schiff base acts as uninegatively charged bidentate chelating agent via the hydroxyl oxygen and azomethine nitrogen atoms. DNA binding studies of all the complexes with calf thymus DNA have been comprehensively investigated using electronic absorption spectroscopy, fluorescence quenching and viscosity studies. The oxidative and photo cleavage affinity of metal complexes towards supercoiled pBR322 DNA has been ascertained by agarose gel electrophoresis assay. From the results, it is observed that all the metal complexes bind effectively to CT-DNA via an intercalative mode of binding and also cleave pBR322 DNA in a promising manner. Further the Cu(II) complexes have shown better binding and cleavage properties towards DNA. The antimicrobial activities of the Schiff bases and their metal complexes were studied on bacterial and fungal strains and the results denoted that the complexes are more potent than their Schiff base ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Directory of Open Access Journals (Sweden)

    Chattopadhyay Debasish

    2009-02-01

    Full Text Available Abstract Background The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. Results We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in

  5. Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Anna Albisetti

    2017-11-01

    Full Text Available Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP but remains attached to the cell body via the flagellum attachment zone (FAZ. The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC circumvents the flagellum. Overlapping the FPC is the hook complex (HC (a sub-structure of the previously named bilobe that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein-FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

  6. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

    Science.gov (United States)

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  7. Zeaxanthin binds to light-harvesting complex stress-related protein to enhance nonphotochemical quenching in Physcomitrella patens.

    Science.gov (United States)

    Pinnola, Alberta; Dall'Osto, Luca; Gerotto, Caterina; Morosinotto, Tomas; Bassi, Roberto; Alboresi, Alessandro

    2013-09-01

    Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)-dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments.

  8. Zeaxanthin Binds to Light-Harvesting Complex Stress-Related Protein to Enhance Nonphotochemical Quenching in Physcomitrella patens[W

    Science.gov (United States)

    Pinnola, Alberta; Dall’Osto, Luca; Gerotto, Caterina; Morosinotto, Tomas; Bassi, Roberto; Alboresi, Alessandro

    2013-01-01

    Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)–dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments. PMID:24014548

  9. The acid-labile subunit of human ternary insulin-like growth factor binding protein complex in serum

    DEFF Research Database (Denmark)

    Juul, A; Møller, S; Mosfeldt-Laursen, E

    1998-01-01

    Circulating insulin-like growth factor-I (IGF-I) is predominantly bound in the trimeric complex comprised of IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS). Circulating concentrations of IGF-I, IGFBP-3 and ALS are believed to reflect the GH secretory status, but the clinical use...... of ALS determination is not known. We therefore, determined the: 1) hepatosplanchnic release of ALS by liver vein catheterization (n=30); 2) 24-h diurnal variation of ALS (n=8); 3) normal age-related ranges of circulating ALS (n=1158); 4) diagnostic value of ALS in 108 patients with childhood-onset GH...... in adults; and 4) ALS levels were below -2 SD in 57 of 79 GHD patients (sensitivity 72%) and above 2 SD in 22 of 29 patients with normal GH response (specificity 76%), which was similar, compared with the diagnostic utility of IGF-I and IGFBP-3. Finally, our findings indicate that hepatic ALS production...

  10. SODs, DNA binding and cleavage studies of new Mn(III) complexes with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol

    Science.gov (United States)

    Shivakumar, L.; Shivaprasad, K.; Revanasiddappa, Hosakere D.

    2013-04-01

    Newly synthesized ligand [2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol] (Bpmp) react with manganese(II) to form mononuclear complexes [Mn(phen)(Bpmp)(CH3COO)(H2O)]·4H2O (1), (phen = 1,10-phenanthroline) and [Mn(Bpmp)2(CH3COO)(H2O)]·5H2O (2). These complexes were characterized by elemental analysis, IR, 1H NMR, Mass, UV-vis spectral studies. Molar conductance and thermogravimetric analysis of these complexes were also recorded. The in vitro SOD mimic activity of Mn(III) complexes were carried out and obtained with good result. The DNA-binding properties of the complexes 1 and 2 were investigated by UV-spectroscopy, fluorescence spectroscopy and viscosity measurements. The spectral results suggest that the complexes 1 and 2 can bind to Calf thymus DNA by intercalation mode. The cleavage properties of these complexes with super coiled pUC19 have been studied using the gel electrophoresis method, wherein both complexes 1 and 2 displayed chemical nuclease activity in the absence and presence of H2O2via an oxidative mechanism. All the complexes inhibit the growth of both Gram positive and Gram negative bacteria to competent level. The MIC was determined by microtiter method.

  11. AFM images of complexes between amylose and Aspergillus niger glucoamylase mutants, native and mutant starch binding domains: a model for the action of glucoamylase

    DEFF Research Database (Denmark)

    Morris, V. M.; Gunning, A. P.; Faults, C. B.

    2005-01-01

    Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild-type A. niger starch binding domains (SBDS), and mutant SBDs (W563K and W590K) lacking either of the two starch...

  12. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

    NARCIS (Netherlands)

    Dessing, Mark C.; Tammaro, Alessandra; Pulskens, Wilco P.; Teske, Gwendoline J.; Butter, Loes M.; Claessen, Nike; van Eijk, Marco; van der Poll, Tom; Vogl, Thomas; Roth, Johannes; Florquin, Sandrine; Leemans, Jaklien C.

    2015-01-01

    Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury.

  13. Crystal Structure of the VapBC Toxin–Antitoxin Complex from Shigella flexneri Reveals a Hetero-Octameric DNA-Binding Assembly

    DEFF Research Database (Denmark)

    Dienemann, Christian; Bøggild, Andreas; Winther, Kristoffer S.

    2011-01-01

    the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total...

  14. Synthesis, molecular docking and DNA binding studies of phthalimide-based copper(II) complex: In vitro antibacterial, hemolytic and antioxidant assessment

    Science.gov (United States)

    Arif, Rizwan; Nayab, Pattan Sirajuddin; Ansari, Istikhar A.; Shahid, M.; Irfan, Mohammad; Alam, Shadab; Abid, Mohammad; Rahisuddin

    2018-05-01

    In the present research work, we prepared N-substituted phthalimide, 2-(-(2-(2-(2-(1,3-dioxoisoindoline-2-yl-ethylamino)ethylamino)ethyl)isoindoline-1,3-dione (DEEI) and its copper(II) complex. The ligand (DEEI) and its Cu(II) complex were structurally identified using absorption, FTIR, NMR, electron spin resonance, X-ray diffraction spectral studies, thermogravimetric and elemental analyses. The electronic spectrum and magnetic moment value proposed that Cu(II) complex has square planar geometry. The DNA interaction ability of the ligand (DEEI) and Cu(II) complex was studied by means of absorption and fluorescence spectrophotometer, viscosity measurements, cyclic voltammetery, and circular dichroism methods. The extent of DNA binding (Kb) with Calf thymus (Ct-DNA) follows the order of Cu(II) complex (1.11 × 106 M-1) > DEEI (1.0 × 105 M-1), indicating that Cu(II) complex interact with Ct-DNA through groove binding mode and more sturdily than ligand (DEEI). Interestingly, in silico predictions were corroborated with in vitro DNA binding studies. The antibacterial evaluation of these compounds was screened against a panel of bacterial strains Pseudomonas aeruginosa (MTCC 2453), Salmonella enterica (MTCC 3224), Streptococcus pneumoniae (MTCC 655), Enterococcus faecalis (MTCC 439), Klebsiella pneumonia and Escherichia coli (ATCC 25922). The results showed that the copper(II) complex has significant antibacterial potential (IC50 = 0.0019 μg/mL) against Salmonella enteric comparable with ligand (DEEI) and standard drug ciprofloxacin. Growth curve study of Cu(II) complex against only three bacterial strains S. enterica, E. faecalis and S. pneumoniae showed its bactericidal nature. Cu(II) complex showed less than 2% hemolysis on human RBCs indicating its non toxic nature. The results of antioxidant assay demonstrated that scavenging activity of Cu(II) complex is higher as compared to ligand and ascorbic acid as standard.

  15. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1987-01-01

    The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more...... than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells......, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only...

  16. Photodynamic effect of light-harvesting, long-lived triplet excited state Ruthenium(II)-polyimine-coumarin complexes: DNA binding, photocleavage and anticancer studies.

    Science.gov (United States)

    Nomula, Raju; Wu, Xueyan; Zhao, Jianzhang; Munirathnam, Nagegownivari R

    2017-10-01

    Two coumarin based Ru II -polyimine complexes (Ru-1 and Ru-2) showing intense absorption of visible light and long-lived triplet excited states (~12-15μs) were used for study of the interaction with DNA. The binding of the complexes with CT-DNA were studied by UV-vis, fluorescence and time-resolved nanosecond transient absorption (ns-TA) spectroscopy. The results suggesting that the complexes interact with CT-DNA by intercalation mode of binding, showing the binding constants (K b ) 6.47×10 4 for Ru-1 and 5.94×10 4 M -1 for Ru-2, in contrast no such results were found for Ru-0. The nanosecond transient absorption spectra of these systems in the presence of CT-DNA showing a clear perturbation in the bleaching region was observed compare to buffer alone. Visible light photoirradiation DNA cleavage was investigated for these complexes by treating with the supercoiled pUC19 DNA and irradiated at 450nm. The reactive species produced upon irradiation of current agents is singlet oxygen ( 1 O 2 ), which results in the generation of other reactive oxygen species (ROS). The complexes shown efficient cleavage activity, converted complete supercoiled DNA to nicked circular at as low as 20μM concentration in 30min of light irradiation time. Significant amount of linear form was generated by Ru-1 at the same conditions. Even though Ru-0 has significant 1 O 2 quantum yield but shown lower cleavage activity compared to other two analogs is due the miserable interaction (binding) with DNA. The cytotoxicity in vitro of the complexes toward HeLa, BEL-7402 and MG-63 cells was assessed by MTT assay. The cellular uptake was observed on BEL-7402 cells under fluorescence microscope. The complexes shown appreciable cytotoxicity towards the cancer cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Spectroscopic investigations of the B12-binding subunit of glutamate mutase: refined solution structure of the complex with the B12-nucleotide, dynamics and binding studies with two corrinoid cofactors

    International Nuclear Information System (INIS)

    Eichmueller, C.

    2002-06-01

    Glutamate mutase is an enzyme isolated from Clostridium tetanomorphum and Clostridium cochlearum. It catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. Coenzyme B12 is required as cofactor for an active enzyme, as the first step of the catalytic cycle is the homolytic cleavage of the cobalt-carbon bond. The rearrangement itself follows a radical mechanism. The holoenzyme is an alpha2beta2 heterotetramer containing two identical catalytic and two B12 binding domains, as well as two coenzyme B12 molecules. The smaller B12 binding domain from Clostridium tetanomorphum, MutS, is known to bind coenzyme B12 in its unusual 'base-off' form. A conserved histidine residue coordinates to the cobalt atom instead of the normally coordinated dimethlybenzimidole in free coenzyme B12. In the present work a refined solution structure of the B12 binding subunit from Clostridium tetanomorphum (MutS) in complex with the detached nucleotide loop of coenzyme B12 has been determined using nuclear magnetic resonance. The found topology is almost identical to the crystal structure of glutamate mutase from C.cochlearum [Reitzer et al., 1999], in contrast to the solution structures obtained for apo-MutS [Hoffmann et al., 2001; Tollinger et al., 1998] and apo-GlmS [Hoffmann et al., 1999]. In these two structures a helix at one side of the B12 nucleotide loop binding pocket is mostly unstructured and shows motions on a microsecond to millisecond timescale. The previously found stabilization of this helix upon B12-nucleotide binding [Tollinger et al., 2001] was confirmed using 13C and 15N labeled MutS. Some differences are found in the structure of the binding pocket and the bound nucleotide loop compared to the crystal structure. This indicates that additional conformational changes occur upon binding of the corrin ring of coenzyme B12. NMR-relaxation measurements performed on apo-MutS showed interesting slow molecular motions not only in the mainly

  18. Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

    International Nuclear Information System (INIS)

    Liu, Zhongchuan; Xie, Tian; Zhong, Qiuping; Wang, Ganggang

    2016-01-01

    The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) in a hole motif has been solved; this novel binding site could be a potential structure-based target for protein engineering of CotA laccase. The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases

  19. Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhongchuan; Xie, Tian [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of (China); Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu 610041, People’s Republic of (China); Zhong, Qiuping [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of (China); Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu 610041, People’s Republic of (China); University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of (China); Wang, Ganggang, E-mail: wanggg@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of (China); Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu 610041, People’s Republic of (China)

    2016-03-24

    The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) in a hole motif has been solved; this novel binding site could be a potential structure-based target for protein engineering of CotA laccase. The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

  20. Tris-amidoximate uranyl complexes via η2 binding mode coordinated in aqueous solution shown by X-ray absorption spectroscopy and density functional theory methods.

    Science.gov (United States)

    Zhang, Linjuan; Qie, Meiying; Su, Jing; Zhang, Shuo; Zhou, Jing; Li, Jiong; Wang, Yu; Yang, Shitong; Wang, Shuao; Li, Jingye; Wu, Guozhong; Wang, Jian Qiang

    2018-03-01

    The present study sheds some light on the long-standing debate concerning the coordination properties between uranyl ions and the amidoxime ligand, which is a key ingredient for achieving efficient extraction of uranium. Using X-ray absorption fine structure combined with theoretical simulation methods, the binding mode and bonding nature of a uranyl-amidoxime complex in aqueous solution were determined for the first time. The results show that in a highly concentrated amidoxime solution the preferred binding mode between UO 2 2+ and the amidoxime ligand is η 2 coordination with tris-amidoximate species. In such a uranyl-amidoximate complex with η 2 binding motif, strong covalent interaction and orbital hybridization between U 5f/6d and (N, O) 2p should be responsible for the excellent binding ability of the amidoximate ligand to uranyl. The study was performed directly in aqueous solution to avoid the possible binding mode differences caused by crystallization of a single-crystal sample. This work also is an example of the simultaneous study of local structure and electronic structure in solution systems using combined diagnostic tools.

  1. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

    2016-04-22

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  2. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    International Nuclear Information System (INIS)

    Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  3. Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Honing, Stephan; Bogdanov, Yury; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Jovanovic, Jasmina N; Pangalos, Menelas N; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2005-10-11

    The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

  4. Host-guest chemistry of dendrimer-drug complexes. 4. An in-depth look into the binding/encapsulation of guanosine monophosphate by dendrimers.

    Science.gov (United States)

    Hu, Jingjing; Fang, Min; Cheng, Yiyun; Zhang, Jiahai; Wu, Qinglin; Xu, Tongwen

    2010-06-03

    In the present study, we investigated the host-guest chemistry of dendrimer/guanosine monophosphate (GMP) and present an in-depth look into the binding/encapsulation of GMP by dendrimers using NMR studies. (1)H NMR spectra showed a significant downfield shift of methylene protons in the outmost layer of the G5 dendrimer, indicating the formation of ion pairs between cationic amine groups of dendrimer and anionic phosphate groups of GMP. Chemical shift titration results showed that the binding constant between G5 dendrimer and GMP is 17,400 M(-1) and each G5 dendrimer has 107 binding sites. The binding of GMP to dendrimers prevents its aggregation in aqueous solutions and thereby enhances its stability. Nuclear Overhauser effect measurements indicated that a GMP binding and encapsulation balance occurs on the surface and in the interior of dendrimer. The binding/encapsulation transitions can be easily tailored by altering the surface and interior charge densities of the dendrimer. All these findings provide a new insight into the host-guest chemistry of dendrimer/guest complexes and may play important roles in the study of dendrimer/DNA aggregates by a "bottom-up" strategy.

  5. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    Science.gov (United States)

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  6. Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

    Science.gov (United States)

    Yu, Zhanyang; Zhang, Yu; Liu, Ning; Yuan, Jing; Lin, Li; Zhuge, Qichuan; Xiao, Jian; Wang, Xiaoying

    2016-07-01

    Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

  7. BINDING OF THE RESPIRATORY CHAIN INHIBITOR ANTIMYCIN TO THE MITOCHONDRIAL bc1 COMPLEX: A NEW CRYSTAL STRUCTURE REVEALS AN ALTERED INTRAMOLECULAR HYDROGEN-BONDING PATTERN.

    OpenAIRE

    Huang, Li-shar; Cobessi, David; Tung, Eric Y.; Berry, Edward A.

    2005-01-01

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity-relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Tw...

  8. Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction

    DEFF Research Database (Denmark)

    Jepsen, H H; Svehag, S E; Jarlbaek, L

    1986-01-01

    showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized...... of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect...

  9. Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig.

    Science.gov (United States)

    Wasilewska, Barbara; Najdzion, Janusz; Równiak, Maciej; Bogus-Nowakowska, Krystyna; Hermanowicz, Beata; Kolenkiewicz, Małgorzata; Żakowski, Witold; Robak, Anna

    2016-03-01

    In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed. Copyright © 2015 Elsevier GmbH. All rights reserved.

  10. DNA binding and cleavage studies of new sulfasalazine-derived dipeptide Zn(II) complex: Validation for specific recognition with 5 Prime -TMP

    Energy Technology Data Exchange (ETDEWEB)

    Tabassum, Sartaj [Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002 (India); Al-Asbahy, Waddhaah M.; Afzal, Mohd.; Shamsi, Manal; Arjmand, Farukh [Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002 (India)

    2012-11-15

    A new water soluble complex [Zn(glygly)(ssz)(H{sub 2}O)]{center_dot}6H{sub 2}O, 1 derived from dipeptide (glycyl glycine) and sulfasalazine was synthesized and characterized by spectroscopic (IR, UV-vis, NMR, ESI-MS) and analytical methods. The in vitro DNA binding studies of complex 1 with calf-thymus DNA were carried out by employing various biophysical methods and molecular docking technique which reveals strong electrostatic binding via phosphate backbone of DNA helix, in addition to partial intercalation. To gain further insight into the molecular recognition at the target site, interaction studies of complex 1 with 5 Prime -TMP and 5 Prime -GMP were carried out by UV-vis titration which was validated by {sup 1}H and {sup 31}P NMR with 5 Prime -TMP, which implicate the preferential selectivity of 1 towards N3 of thymine. Complex 1 is accessible to minor groove of DNA and cleaved pBR322 DNA via hydrolytic pathway (validated by T4 ligase assay). - Graphical abstract: Synthesis, characterization, DNA binding and cleavage studies of [Zn(glygly)(ssz)(H{sub 2}O)]{center_dot}6H{sub 2}O (1) containing glycyl glycine and sulfasalazine ligand. Complex 1 recognize minor groove of DNA and show hydrolytic DNA cleavage. Highlights: Black-Right-Pointing-Pointer Novel Zn(II) complex 1 bearing bioactive glycyl glycine and sulfasalazine ligand scaffold. Black-Right-Pointing-Pointer Cleavage activity of 1 was enhanced in presence of activators: H{sub 2}O{sub 2}>MPA>GSH>Asc. Black-Right-Pointing-Pointer Complex 1 recognize minor groove as depicted in the cleavage pattern and molecular docking. Black-Right-Pointing-Pointer Complex 1 cleaves pBR322 DNA via hydrolytic mechanism and validated by T4 DNA ligase experiments.

  11. DNA binding and cleavage studies of new sulfasalazine-derived dipeptide Zn(II) complex: Validation for specific recognition with 5′–TMP

    International Nuclear Information System (INIS)

    Tabassum, Sartaj; Al–Asbahy, Waddhaah M.; Afzal, Mohd.; Shamsi, Manal; Arjmand, Farukh

    2012-01-01

    A new water soluble complex [Zn(glygly)(ssz)(H 2 O)]·6H 2 O, 1 derived from dipeptide (glycyl glycine) and sulfasalazine was synthesized and characterized by spectroscopic (IR, UV–vis, NMR, ESI–MS) and analytical methods. The in vitro DNA binding studies of complex 1 with calf–thymus DNA were carried out by employing various biophysical methods and molecular docking technique which reveals strong electrostatic binding via phosphate backbone of DNA helix, in addition to partial intercalation. To gain further insight into the molecular recognition at the target site, interaction studies of complex 1 with 5′-TMP and 5′-GMP were carried out by UV–vis titration which was validated by 1 H and 31 P NMR with 5′-TMP, which implicate the preferential selectivity of 1 towards N3 of thymine. Complex 1 is accessible to minor groove of DNA and cleaved pBR322 DNA via hydrolytic pathway (validated by T4 ligase assay). - Graphical abstract: Synthesis, characterization, DNA binding and cleavage studies of [Zn(glygly)(ssz)(H 2 O)]·6H 2 O (1) containing glycyl glycine and sulfasalazine ligand. Complex 1 recognize minor groove of DNA and show hydrolytic DNA cleavage. Highlights: ► Novel Zn(II) complex 1 bearing bioactive glycyl glycine and sulfasalazine ligand scaffold. ► Cleavage activity of 1 was enhanced in presence of activators: H 2 O 2 >MPA>GSH>Asc. ► Complex 1 recognize minor groove as depicted in the cleavage pattern and molecular docking. ► Complex 1 cleaves pBR322 DNA via hydrolytic mechanism and validated by T4 DNA ligase experiments.

  12. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with /sup 125/I-labelled Clq binding and Raji-cell RIA tests

    Energy Technology Data Exchange (ETDEWEB)

    Casali, P; Bossus, A; Carpentier, N A; Lambert, P H [Hopital Cantonal Geneve (Switzerland)

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 ..mu..g/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 ..mu..g/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the /sup 125/I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, /sup 125/I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.

  13. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex

    Science.gov (United States)

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy = 2,2-bipyridine, Hbcpip = 2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5 ± 1.4) × 105 M-1 in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  14. Synthesis and characterization of mononuclear copper(II complex of tetradentate N2S2 donor set and the study of DNA and bovine serum albumin binding

    Directory of Open Access Journals (Sweden)

    Sandipan Sarkar

    2014-12-01

    Full Text Available One mononuclear copper(II complex, containing neutral tetradentate NSSN-type ligands, of formulation [Cu II(L 1Cl]ClO 4 (1, was synthesized and isolated in pure form [where L 1˭ 1,3-bis(3-pyridylmethylthiopropane]. Green-colored copper(II complex was characterized by physicochemical, spectroscopic methods and conductivity measurement. These experimental data matched well with the proposed structure of the complex. Biological activity of the complex (1 toward calf thymus DNA and bovine serum albumin has been examined systematically and groove-binding behavior of the Copper(II complex 1 with calf thymus DNA has been observed from the spectral study.

  15. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    International Nuclear Information System (INIS)

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  16. Factors affecting nucleolytic efficiency of some ternary metal complexes with DNA binding and recognition domains. Crystal and molecular structure of Zn(phen)(edda).

    Science.gov (United States)

    Seng, Hoi-Ling; Ong, Han-Kiat Alan; Rahman, Raja Noor Zaliha Raja Abd; Yamin, Bohari M; Tiekink, Edward R T; Tan, Kong Wai; Maah, Mohd Jamil; Caracelli, Ignez; Ng, Chew Hee

    2008-11-01

    The binding selectivity of the M(phen)(edda) (M=Cu, Co, Ni, Zn; phen=1,10-phenanthroline, edda=ethylenediaminediacetic acid) complexes towards ds(CG)(6), ds(AT)(6) and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(II) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N(4)O(2) octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that inter-digitate with adjacent double layers via pi...pi interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling.

  17. CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

    Science.gov (United States)

    Horejsí, Zuzana; Takai, Hiroyuki; Adelman, Carrie A; Collis, Spencer J; Flynn, Helen; Maslen, Sarah; Skehel, J Mark; de Lange, Titia; Boulton, Simon J

    2010-09-24

    TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

    Science.gov (United States)

    Bazzicalupi, Carla; Ferraroni, Marta; Papi, Francesco; Massai, Lara; Bertrand, Benoît; Messori, Luigi; Gratteri, Paola; Casini, Angela

    2016-03-18

    The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. DNA-Binding Studies of Some Potential Antitumor 2,2'-bipyridine Pt(II)/Pd(II) Complexes of piperidinedithiocarbamate. Their Synthesis, Spectroscopy and Cytotoxicity.

    Science.gov (United States)

    Mansouri-Torshizi, Hassan; Eslami-Moghadam, Mahboube; Divsalar, Adeleh; Saboury, Ali-Akbar

    2011-12-01

    In this study two platinum(II) and palladium(II) complexes of the type [M(bpy)(pip-dtc)]NO3 (where M=Pt(II) or Pd(II), bpy=2,2'-bipyridine, pip-dtc=piperidinedithiocarbamate) were synthesized by reaction between diaquo-2,2'-bipyridine Pt(II)/Pd(II) nitrate and sodium salt of dithiocarbamate. These cationic water soluble complexes were characterized by elemental analysis, molar conductance, IR, electronic and 1H NMR spectroscopic studies. The cyclic dithiocarbamate was found to coordinate as bidentate fasion with Pt(II) or Pd(II) center. Their biological activities were tested against chronic myelogenous leukemia cell line, K562, at micromolar concentration. The obtained cytotoxic concentration (IC50) values were much lower than cisplatin. The interaction of these complexes with highly polymerized calf thymus DNA (ct-DNA) was extensively studied by means of electronic absorption, fluorescence, circular dichroism and other measurements. The experimental results, thermodynamic and binding parameters, suggested that these complexes cooperatively bind to DNA presumably via intercalation. Moreover, the tendency of the Pt(II) complex to interact with DNA was more than that of Pd(II) complex.

  20. Structure of a Glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface

    Science.gov (United States)

    Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.; Hammel, Michal; Lambert, Lester J.; Waddell, M. Brett; Mittag, Tanja; DeCaprio, James A.; Schulman, Brenda A.

    2012-01-01

    Summary The ~300 human Cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1’s RING domain, regulates the RBX1-CUL1-containing SCFFBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN’s selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition. PMID:22748924

  1. Structures of the APC–ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners

    Science.gov (United States)

    Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

    2015-01-01

    The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC–Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC–ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC–ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC–ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC–ARM binding partners. PMID:27462415

  2. Structures of the APC-ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners.

    Science.gov (United States)

    Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

    2015-01-01

    The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC-Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC-ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC-ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC-ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC-ARM binding partners.

  3. Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding

    Directory of Open Access Journals (Sweden)

    Robert Gustafsson

    2018-04-01

    Full Text Available Botulinum neurotoxins (BoNTs are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G. Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C as their protein receptors. We here present a high resolution (2.0 Å co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.

  4. Crystal structure of the high-affinity Na+K+-ATPase-ouabain complex with Mg2+ bound in the cation binding site.

    Science.gov (United States)

    Laursen, Mette; Yatime, Laure; Nissen, Poul; Fedosova, Natalya U

    2013-07-02

    The Na(+),K(+)-ATPase maintains electrochemical gradients for Na(+) and K(+) that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na(+),K(+)-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na(+),K(+)-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg(2+). K(+) binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.

  5. GUN4-Porphyrin Complexes Bind the ChlH/GUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis[W

    Science.gov (United States)

    Adhikari, Neil D.; Froehlich, John E.; Strand, Deserah D.; Buck, Stephanie M.; Kramer, David M.; Larkin, Robert M.

    2011-01-01

    The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O2. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex. PMID:21467578

  6. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)

    2012-02-21

    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  7. The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action

    Energy Technology Data Exchange (ETDEWEB)

    Band, L.; Xu, Heng; Bykov, V.; Rothman, R.B.; Kim, Chongho; Newman, A.; Jacobson, A.E.; Rice, K.C. (NIDDK, Bethesda, MD (USA)); Greig, N. (NIA, Bethesda, MD (USA))

    1990-01-01

    The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl ((+)-cis-MF), followed by extensive washing of the membranes, produces a wash-resistant decreasing in the binding of ({sup 3}H)-(D-ala{sup 2}, D-leu{sup 5})enkephalin to the d binding site of the opioid receptor complex ({delta}{sub cx} binding site). Intravenous administration of (+)-cis-MF (50 {mu}g/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of {delta}{sub cx} and {mu} binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6{beta}-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked {delta}{sub cx} binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.

  8. Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site.

    Directory of Open Access Journals (Sweden)

    Hua Yang

    2010-09-01

    Full Text Available Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107, including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb. Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (alpha2-3 receptor binding profile, with only moderate binding to human-type (alpha2-6 receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  9. Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria.

    Science.gov (United States)

    Ranganathan, Sridevi; Cheung, Jonah; Cassidy, Michael; Ginter, Christopher; Pata, Janice D; McDonough, Kathleen A

    2018-01-09

    Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr's DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr's multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex*

    Science.gov (United States)

    Douglas, Max E.

    2016-01-01

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (“G1-like”) and high affinity recruitment when CMG assembly takes place (“S-phase-like”). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. PMID:26719337

  11. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex.

    Science.gov (United States)

    Douglas, Max E; Diffley, John F X

    2016-03-11

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1

    International Nuclear Information System (INIS)

    Meulenbroek, Elisabeth M.; Pannu, Navraj S.

    2011-01-01

    Human Cockayne syndrome protein A has been cocrystallized with human DNA damage-binding protein 1 and data have been collected to 2.9 Å resolution. Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility

  13. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

    Science.gov (United States)

    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  14. A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Azad A Sulaiman

    2016-11-01

    Full Text Available The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs in their evasion of antibody-dependent cell cytotoxicity (ADCC by reducing the NO response of macrophages-again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow

  15. Three-dimensional structure of the ligand-binding core of GluR2 in complex with the agonist (S)-ATPA

    DEFF Research Database (Denmark)

    Lunn, Marie-Louise; Hogner, Anders; Stensbøl, Tine B

    2003-01-01

    Two X-ray structures of the GluR2 ligand-binding core in complex with (S)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid ((S)-ATPA) have been determined with and without Zn(2+) ions. (S)-ATPA induces a domain closure of ca. 21 degrees compared to the apo form. The tert-butyl moiety...

  16. Human T-lymphotropic virus type 1 Tax protein complexes with P-TEFb and competes for Brd4 and 7SK snRNP/HEXIM1 binding.

    Science.gov (United States)

    Cho, Won-Kyung; Jang, Moon Kyoo; Huang, Keven; Pise-Masison, Cynthia A; Brady, John N

    2010-12-01

    Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.

  17. Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern.

    Science.gov (United States)

    Huang, Li-Shar; Cobessi, David; Tung, Eric Y; Berry, Edward A

    2005-08-19

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.

  18. Binding of hydrocarbons and other extremely weak ligands to transition metal complexes that coordinate hydrogen: Investigation of cis-interactions and delocalized bonding involving sigma bonds

    International Nuclear Information System (INIS)

    Kubas, G.J.; Eckert, J.; Luo, X.L.

    1997-01-01

    This is the final report of a three-year Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). At the forefront of chemistry are efforts to catalytically transform the inert C-H bonds in alkanes to more useful products using metal compounds. The goal is to observe binding and cleavage of alkane C-H bonds on metals or to use related silane Si-H bonding as models, analogous to the discovery of hydrogen (H 2 ) binding to metals. Studies of these unique sigma complexes (M hor-ellipsis H-Y; Y double-bond H, Si, C) will aid in developing new catalysts or technologies relevant to DOE interest, e.g., new methods for tritium isotope separation. Several transition metals (Mo, W, Mn, and Pt) were found to reversibly bind and cleave H 2 , silanes, and halocarbons. The first metal-SiH 4 complexes, thus serving as a model for methane reactions. A second goal is to study the dynamics and energetics of H-Y bonds on metals by neutron scattering, and evidence for interactions between bound H-Y and nearby H atoms on metal complexes has been found

  19. Formation of Mixed-Ligand Complexes of Pd2+ with Nucleoside 5'-Monophosphates and Some Metal-Ion-Binding Nucleoside Surrogates

    Directory of Open Access Journals (Sweden)

    Oleg Golubev

    2014-10-01

    Full Text Available Formation of mixed-ligand Pd2+ complexes between canonical nucleoside 5'-monophosphates and five metal-ion-binding nucleoside analogs has been studied by 1H-NMR spectroscopy to test the ability of these nucleoside surrogates to discriminate between unmodified nucleobases by Pd2+-mediated base pairing. The nucleoside analogs studied included 2,6-bis(3,5-dimethylpyrazol-1-yl-, 2,6-bis(1-methylhydrazinyl- and 6-(3,5-dimethylpyrazol-1-yl-substituted 9-(β-d-ribofuranosylpurines 1–3, and 2,4-bis(3,5-dimethylpyrazol-1-yl- and 2,4-bis(1-methylhydrazinyl-substituted 5-(β-d-ribofuranosyl-pyrimidines 4–5. Among these, the purine derivatives 1-3 bound Pd2+ much more tightly than the pyrimidine derivatives 4, 5 despite apparently similar structures of the potential coordination sites. Compounds 1 and 2 formed markedly stable mixed-ligand Pd2+ complexes with UMP and GMP, UMP binding favored by 1 and GMP by 2. With 3, formation of mixed-ligand complexes was retarded by binding of two molecules of 3 to Pd2+.

  20. Binding of the Respiratory Chain Inhibitor Antimycin to theMitochondrial bc1 Complex: A New Crystal Structure Reveals an AlteredIntramolecular Hydrogen-Bonding Pattern

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Cobessi, David; Tung, Eric Y.; Berry, Edward A.

    2005-05-10

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex.Structure-activity-relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28Angstrom resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cyt b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alpha-A helix.

  1. Studies on the pathogenesis of Aleutian disease of mink. X. demonstration of immune complexes by the /sup 125/I-C 1 q binding test after experimental infection

    Energy Technology Data Exchange (ETDEWEB)

    Mueller-Peddinghaus, R [Kali-Chemie Pharma G.m.b.H., Hannover (Germany, F.R.). Abt. fuer Experimentelle Pathologie; Meyer zu Schwabedissen, H [Medizinische Hochschule Hannover (Germany, F.R.). Abt. fuer Klinische Immunologie und Bluttransfusionswesen; Kalden, J R [Erlangen-Nuernberg Univ., Erlangen (Germany, F.R.). Inst. und Poliklinik fuer Klinische Immunologie; Trautwein, G; Ueberschaer, S [Tieraerztliche Hochschule Hannover (Germany, F.R.). Inst. fuer Pathologie

    1980-01-01

    Aleutian disease (AD) of mink most closely resembles systemic lupus erythematosus (SLE) in man; both are immune complex disease. In experimental AD serum immune complexes are determined by the /sup 125/J-C 1 q-binding test using human C 1 q. Mink (n = 12) infected intraperitoneally with Aleutian disease virus (ADV), grown in fetal mink kidney cells, developed during the course of infection a mean of /sup 125/I-C 1 q serum binding equivalent to 3.62 +- 1.68 mg./ml. aggr. HGG. (aggregated human immunoglobulin). Sera of mink (n = 8) which were infected with ADV grown in L-cells showed a less marked /sup 125/I-C 1 q binding with a mean equivalent to 2.52 +- 1.43 mg./ml. aggr. HGG. In contrast control animals (n = 8) treated with non-ADV-infected mink epidermal fibroblasts or Eagle's minimal essential medium substituted with fetal calf serum only bound /sup 125/I-C 1 q equivalent to 1.02 +- 0.99 mg./ml. aggr. HGG. In mink infected with ADV propagated in fetal mink kidney cells a constant increase in the /sup 125/I-C 1 q serum binding occurred from the 4th to the 7th and 13th week after ADV infection. Mink which were infected with ADV propagated in mouse L-cells exhibited a different pattern of the /sup 125/I-C 1 q serum binding capacity with a sharp increase from the 4th to the 7th week, followed by a decline towards the 13th week post infection. The serum /sup 125/I-C 1 q binding capacity of all experimental animal groups exhibited at different times of the experiment a significant correlation with the presence of hypergammaglobulinaemia and raised ADV-antibody titers. From the data obtained it appears that the /sup 125/I-C 1 q binding test, utilizing human C 1 q, is a suitable method for the detection of circulating serum immune complexes in mink during the course of ADV-infection.

  2. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity-labeling

    International Nuclear Information System (INIS)

    Baxter, R.C.; Martin, J.L.

    1989-01-01

    To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit

  3. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  4. Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2016-05-28

    The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.

  5. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish; (UAB)

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate

  6. Synthesis, spectroscopic studies, DFT calculations, electrochemical evaluation, BSA binding and molecular docking of an aroylhydrazone -based cis-dioxido Mo(VI) complex

    Science.gov (United States)

    Mohamadi, Maryam; Faghih-Mirzaei, Ehsan; Ebrahimipour, S. Yousef; Sheikhshoaie, Iran; Haase, Wolfgang; Foro, Sabine

    2017-07-01

    A cis-dioxido Mo(VI) complex, [MoO2(L)(MeOH)], [L2-: (3-methoxy-2-oxidobenzylidene) benzohydrazonate], has been synthesized and characterized using physicochemical and spectroscopic techniques including elemental analysis, FT-IR, 1HNMR, UV-Vis spectroscopy, molar conductivity and single crystal X-ray diffraction. DFT calculations in the ground state of the complex were carried out using hybrid functional B3LYP with DGDZVP as basis set. Non-linear optical properties including electric dipole moment (μ), polarizability (α) and molecular first hyperpolarizability (β) of the compound were also computed. The values of linear polarizability and first hyperpolarizability obtained for the studied molecule indicated that the compound could be a good candidate of nonlinear optical materials. TD-DFT calculation and molecular electrostatic potential (MEP) were also performed. The thermodynamic properties (heat capacity, entropy, and enthalpy) of the complex at different temperatures have been calculated. The interaction of a synthesized complex, with bovine serum albumin was also thoroughly investigated using experimental and theoretical studies. UV-Vis absorption and fluorescence quenching techniques were used to determine the binding parameters as well as the mechanism of the interaction. The values of binding constants were in the range of 104-105 M-1 demonstrating a moderate interaction between the synthesized complex and BSA making the protein suitable for transportation and delivery of the compound. Thermodynamic parameters were also indicating a binding through van der Waals force or hydrogen bond of [MoO2(L)(MeOH)] to BSA. The results obtained from docking studies were consistent to those obtained from experimental studies.

  7. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test

    International Nuclear Information System (INIS)

    Casali, P.; Bossus, A.; Carpentier, N.A.; Lambert, P.H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125 I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125 I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. (author)

  8. Structure of the lutein-binding domain of human StARD3 at 1.74 Å resolution and model of a complex with lutein

    Energy Technology Data Exchange (ETDEWEB)

    Horvath, Martin P., E-mail: martin.horvath@utah.edu; George, Evan W.; Tran, Quang T.; Baumgardner, Kody; Zharov, Gabe; Lee, Sarah [University of Utah, 257 S 1400 E, Salt Lake City, UT 84112 (United States); Sharifzadeh, Hassan; Shihab, Saeed; Mattinson, Ty; Li, Binxing; Bernstein, Paul S., E-mail: martin.horvath@utah.edu [Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT 84132 (United States)

    2016-07-27

    The structure of a START-domain protein known to bind lutein in the human retina is reported to an improved resolution limit. Rigid-body docking demonstrates that at least a portion of lutein must protrude from the large tunnel-like cavity characteristic of this helix-grip protein and suggests a mechanism for lutein binding specificity. A crystal structure of the lutein-binding domain of human StARD3 (StAR-related lipid-transfer protein 3; also known as MLN64) has been refined to 1.74 Å resolution. A previous structure of the same protein determined to 2.2 Å resolution highlighted homology with StARD1 and shared cholesterol-binding character. StARD3 has since been recognized as a carotenoid-binding protein in the primate retina, where its biochemical function of binding lutein with specificity appears to be well suited to recruit this photoprotective molecule. The current and previous structures correspond closely to each other (r.m.s.d. of 0.25 Å), especially in terms of the helix-grip fold constructed around a solvent-filled cavity. Regions of interest were defined with alternate conformations in the current higher-resolution structure, including Arg351 found within the cavity and Ω1, a loop of four residues found just outside the cavity entrance. Models of the complex with lutein generated by rigid-body docking indicate that one of the ionone rings must protrude outside the cavity, and this insight has implications for molecular interactions with transport proteins and enzymes that act on lutein. Interestingly, models with the ∊-ionone ring characteristic of lutein pointing towards the bottom of the cavity were associated with fewer steric clashes, suggesting that steric complementarity and ligand asymmetry may play a role in discriminating lutein from the other ocular carotenoids zeaxanthin and meso-zeaxanthin, which only have β-ionone rings.

  9. The structure and binding energy of K+endash ether complexes: A comparison of MP2, RI-MP2, and density functional methods

    International Nuclear Information System (INIS)

    Feller, D.; Apra, E.; Nichols, J.A.; Bernholdt, D.E.

    1996-01-01

    The structures and binding energies of several cation:ether complexes (K + :dimethyl ether, K + :dimethoxyethane, K + :12-crown-4 and K + :18-crown-6) were determined with second and fourth order perturbation theory using correlation consistent basis sets. Several of these are the largest correlated calculations yet attempted on crown ethers. The observed systematic convergence to the complete basis set limit provides a standard by which the accuracy of previous studies can be measured and facilitates the calibration of density functional methods. Recent Fouier transform ion cyclotron resonance experiments predicted K + :18-crown-6 binding energies which were significantly smaller than ab initio calculations. None of the potential sources of error examined in the present study were large enough to explain this difference. Although the 6-31+G* basis set used in an earlier theoretical study was smaller than the smallest of the correlation consistent basis sets, with suitable correction for basis set superposition error, it appears capable of yielding binding energies within several kcal/mol of the basis set limit. Perturbation theory calculations exploiting the open-quote open-quote resolution of the identity close-quote close-quote approximation were found to faithfully reproduce binding energies and conformational differences. Although the cation endash ether interaction is dominated by classical electrostatics, the accuracy of density functional techniques was found to be quite sensitive to the choice of functionals. The local density SVWN procedure performed well for binding energies and conformational differences, while underestimating K + O distances by up to 0.08 A. The gradient-corrected Becke endash Lee endash Yang endash Parr functional underestimated the K + :12c4 binding energy by 4 endash 7 kcal/mol or 15%. copyright 1996 American Institute of Physics

  10. An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

    Science.gov (United States)

    Gianti, Eleonora; Zauhar, Randy J.

    2015-05-01

    The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

  11. High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

    Science.gov (United States)

    Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2018-05-01

    K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. An SH2 domain model of STAT5 in complex with phospho-peptides define "STAT5 Binding Signatures".

    Science.gov (United States)

    Gianti, Eleonora; Zauhar, Randy J

    2015-05-01

    The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

  13. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-01-01

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

  14. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-04-15

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer

    International Nuclear Information System (INIS)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-01-01

    The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms. The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis

  16. Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2

    International Nuclear Information System (INIS)

    Ivancic, Monika; Daly, Roger J.; Lyons, Barbara A.

    2003-01-01

    The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central β-sheet capped at each end by an α-helix. The presence of a four residue insertion in the region between β-strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a β-turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed

  17. MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

    Directory of Open Access Journals (Sweden)

    Kaori Asamitsu

    Full Text Available In this study, we applied molecular dynamics (MD simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs, lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

  18. The DNA binding site specificity and antiproliferative property of ternary Pt(II) and Zn(II) complexes of phenanthroline and N,N'-ethylenediaminediacetic acid.

    Science.gov (United States)

    Nakamura, Yusuke; Taruno, Yoko; Sugimoto, Masashi; Kitamura, Yusuke; Seng, Hoi Ling; Kong, Siew Ming; Ng, Chew Hee; Chikira, Makoto

    2013-03-14

    The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

  19. Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

    International Nuclear Information System (INIS)

    Wang Yonggang; Nakashima, Nobutaka; Sekiguchi, Takeshi; Nishimoto, Takeharu

    2005-01-01

    A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2Δ gtr1Δ gtr2Δ was lethal, while a double mutant: gtr1Δ gtr2Δ survived well, indicating that Yrb2p protected cells from the killing effect of gtr1Δ gtr2Δ. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p

  20. Module structure of interphotoreceptor retinoid-binding protein (IRBP may provide bases for its complex role in the visual cycle – structure/function study of Xenopus IRBP

    Directory of Open Access Journals (Sweden)

    Ghosh Debashis

    2007-08-01

    . However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules. Conclusion Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.

  1. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T. (AS); (UTSMC)

    2012-02-27

    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  2. New transition metal complexes of 2,4-dihydroxybenzaldehyde benzoylhydrazone Schiff base (H2dhbh): Synthesis, spectroscopic characterization, DNA binding/cleavage and antioxidant activity

    Science.gov (United States)

    Aboafia, Seyada A.; Elsayed, Shadia A.; El-Sayed, Ahmed K. A.; El-Hendawy, Ahmed M.

    2018-04-01

    New complexes [VO2(Hdhbh)] (1), [VO(phen)(dhbh)].1.5H2O (2), [Zn(Hdhbh)2] (3), [MoO2(dhbh)(D)] (D = H2O (4) or MeOH (5)), [Ru(PPh3)(dhbh)Cl(H2O)] (6), and [Pd(Hdhbh)Cl]·H2O (7) (H2dhbh = Schiff base derived from 2,4-dihydroxybenzaldehyde and benzoylhydrazone) have been isolated and characterized by IR, 1H NMR, Mass, UV-Visible and ESR spectroscopy. They were also investigated by cyclic voltammetry, thermal and magnetic measurements and the structure of complex cis-[MoO2(dhbh)(H2O)] (4) was solved by X-ray crystallography. Analytical data showed that H2dhbh behaves as monobasic/or dibasic tridentate ligand via phenolate O, azomethine N and amide O/or deprotonated amide O atoms. Antioxidant activity of the complexes has been evaluated against DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and it has been found that oxovandium (IV) complex (2) displays the highest radical scavenging potency comparable to ascorbic acid as a standard antioxidant. The DNA binding properties of the ligand and its complexes have been investigated by electronic spectroscopy together with DNA cleavage by gel electrophoresis whose results showed also that vanadium (IV) complex (2) has a significant oxidative cleavage among other complexes.

  3. DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

    Science.gov (United States)

    Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

    2013-02-01

    Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

  4. The structure of an LIM-only protein 4 (LMO4 and Deformed epidermal autoregulatory factor-1 (DEAF1 complex reveals a common mode of binding to LMO4.

    Directory of Open Access Journals (Sweden)

    Soumya Joseph

    Full Text Available LIM-domain only protein 4 (LMO4 is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1, with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1 and C-terminal binding protein interacting protein (CtIP/RBBP8. Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.

  5. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding.

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  6. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  7. Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

    DEFF Research Database (Denmark)

    Pedersen, Natasja Wulff; Chandran, P. Anoop; Qian, Yu

    2017-01-01

    Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide...... automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore...... laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0...

  8. Impact of aromaticity on anticancer activity of polypyridyl ruthenium(II) complexes: synthesis, structure, DNA/protein binding, lipophilicity and anticancer activity.

    Science.gov (United States)

    Čanović, Petar; Simović, Ana Rilak; Radisavljević, Snežana; Bratsos, Ioannis; Demitri, Nicola; Mitrović, Marina; Zelen, Ivanka; Bugarčić, Živadin D

    2017-10-01

    With the aim of assessing how the aromaticity of the inert chelating ligand can influence the activity of ruthenium(II) polypyridyl complexes, two new monofunctional ruthenium(II) complexes, [Ru(Cl-Ph-tpy)(phen)Cl]Cl (1) and [Ru(Cl-Ph-tpy)(o-bqdi)Cl]Cl (2) (where Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2″-terpyridine, phen = 1,10-phenanthroline, o-bqdi = o-benzoquinonediimine), were synthesized. All complexes were fully characterized by elemental analysis and spectroscopic techniques (IR, UV-Vis, 1D and 2D NMR, XRD). Their chemical behavior in aqueous solution was studied by UV-Vis and NMR spectroscopy showing that both compounds are relatively labile leading to the formation of the corresponding aqua species 1a and 2a. 1 H NMR spectroscopy studies performed on complexes 1 and 2 demonstrated that after the hydrolysis of the Cl ligand, they are capable to interact with guanine derivatives (i.e., 9-methylguanine (9MeG) and 5'-GMP) through the N7, forming monofunctional adduct. The kinetics and the mechanism of the reaction of complexes 1 and 2 with the biologically more relevant 5'-GMP ligand were studied by UV-Vis spectroscopy. DNA/protein interactions of the complexes have been examined by photophysical studies, which demonstrated a bifunctional binding mode of the complexes with DNA and the complexes strongly quench the fluorescence intensity of bovine serum albumin (BSA) through the mechanism of both static and dynamic quenching. Complexes 1 and 2 strongly induced apoptosis of treated cancer cells with high percentages of apoptotic cells and negligible percentage of necrotic cells. In addition, both ruthenium complexes decreased Bcl-2/Bax ratio causing cytochrome c mitochondrial release, the activation of caspase-3 and induction of apoptosis.

  9. Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

    International Nuclear Information System (INIS)

    Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

    1987-01-01

    In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

  10. Formation of a dinitrosyl iron complex by NorA, a nitric oxide-binding di-iron protein from Ralstonia eutropha H16.

    Science.gov (United States)

    Strube, Katja; de Vries, Simon; Cramm, Rainer

    2007-07-13

    In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Diferrous NorA binds two molecules of NO maximally. Unexpectedly, binding of NO to the diferrous NorA required an external reductant. Two different NorA-NO species could be resolved. A minor species (up to 20%) showed an S = (1/2) EPR signal with g( perpendicular) = 2.041, and g( parallel) = 2.018, typical of a paramagnetic dinitrosyl iron complex. The major species was EPR-silent, showing characteristic signals at 420 nm and 750 nm in the optical spectrum. This species is proposed to represent a novel dinitrosyl iron complex of the form Fe(2+)-[NO](2)(2-), i.e. NO is bound as NO(-). The NO binding capacity of NorA in conjunction with its high cytoplasmic concentration (20 mum) suggests that NorA regulates transcription by lowering the free cytoplasmic concentration of NO.

  11. Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

    Science.gov (United States)

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Ogorzalek Loo, Rachel R.; Loo, Joseph A.

    2014-12-01

    "Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

  12. Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with beta-cyclodextrin by affinity capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Mikysková, Hana; Kaiser, Martin Maxmilian; Janeba, Zlatko; Holý, Antonín; Kašička, Václav

    2016-01-01

    Roč. 37, č. 2 (2016), s. 239-247 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA13-17224S; GA ČR(CZ) GA15-01948S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * affinity capillary electrophoresis * binding constant * nucleotide analogs * beta-cyclodextrin Subject RIV: CB - Analytical Chemistry , Separation Impact factor: 2.744, year: 2016

  13. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha; Hamdan, Samir; Richardson, Charles C.

    2010-01-01

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Predicting binding poses and affinities for protein - ligand complexes in the 2015 D3R Grand Challenge using a physical model with a statistical parameter estimation

    Science.gov (United States)

    Grudinin, Sergei; Kadukova, Maria; Eisenbarth, Andreas; Marillet, Simon; Cazals, Frédéric

    2016-09-01

    The 2015 D3R Grand Challenge provided an opportunity to test our new model for the binding free energy of small molecules, as well as to assess our protocol to predict binding poses for protein-ligand complexes. Our pose predictions were ranked 3-9 for the HSP90 dataset, depending on the assessment metric. For the MAP4K dataset the ranks are very dispersed and equal to 2-35, depending on the assessment metric, which does not provide any insight into the accuracy of the method. The main success of our pose prediction protocol was the re-scoring stage using the recently developed Convex-PL potential. We make a thorough analysis of our docking predictions made with AutoDock Vina and discuss the effect of the choice of rigid receptor templates, the number of flexible residues in the binding pocket, the binding pocket size, and the benefits of re-scoring. However, the main challenge was to predict experimentally determined binding affinities for two blind test sets. Our affinity prediction model consisted of two terms, a pairwise-additive enthalpy, and a non pairwise-additive entropy. We trained the free parameters of the model with a regularized regression using affinity and structural data from the PDBBind database. Our model performed very well on the training set, however, failed on the two test sets. We explain the drawback and pitfalls of our model, in particular in terms of relative coverage of the test set by the training set and missed dynamical properties from crystal structures, and discuss different routes to improve it.

  15. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha

    2010-04-06

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  17. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA

    International Nuclear Information System (INIS)

    Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2007-01-01

    The mRNA-binding domain of E. coli selenocysteine-specific elongation factor SelB (residues 478–614; SelB-WH3/4) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was prepared by in vitro transcription. The purified SelB-WH3/4–SECIS RNA complex crystallized in space group C2 and diffracted to 2.3 Å. In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA Sec to the ribosomal A site. The minimum C-terminal fragment (residues 478–614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 Å resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 Å. The asymmetric unit contains one WH3/4-domain–RNA complex. The Matthews coefficient was calculated to be 3.37 Å 3 Da −1 and the solvent content was estimated to be 67.4%

  18. Microwave-Assisted Synthesis of Arene Ru(II Complexes Induce Tumor Cell Apoptosis Through Selectively Binding and Stabilizing bcl-2 G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Yanhua Chen

    2016-05-01

    Full Text Available A series of arene Ru(II complexes coordinated with phenanthroimidazole derivatives, [(η6-C6H6Ru(lCl]Cl(1b L = p-ClPIP = 2-(4-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 2b L = m-ClPIP = 2-(3-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 3b L = p-NPIP = 2-(4-Nitrophenylimidazole[4,5f] 1,10-phenanthroline; 4b L = m-NPIP = 2-(3-Nitrophenyl imidazole [4,5f] 1,10-phenanthroline were synthesized in yields of 89.9%–92.7% under conditions of microwave irradiation heating for 30 min to liberate four arene Ru(II complexes (1b, 2b, 3b, 4b. The anti-tumor activity of 1b against various tumor cells was evaluated by MTT assay. The results indicated that this complex blocked the growth of human lung adenocarcinoma A549 cells with an IC50 of 16.59 μM. Flow cytometric analysis showed that apoptosis of A549 cells was observed following treatment with 1b. Furthermore, the in vitro DNA-binding behaviors that were confirmed by spectroscopy indicated that 1b could selectively bind and stabilize bcl-2 G-quadruplex DNA to induce apoptosis of A549 cells. Therefore, the synthesized 1b has impressive bcl-2 G-quadruplex DNA-binding and stabilizing activities with potential applications in cancer chemotherapy.

  19. N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

    Science.gov (United States)

    Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas; Mizuguchi, Gaku; Wei, Debbie; Xiao, Hua; Luk, Ed; Ranjan, Anand; Wu, Carl

    2009-03-06

    Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

  20. Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites.

    OpenAIRE

    Kim, M J; Yamamoto, D; Matsumoto, K; Inoue, M; Ishida, T; Mizuno, H; Sumiya, S; Kitamura, K

    1992-01-01

    In order to investigate the binding mode of E64-c (a synthetic cysteine proteinase inhibitor) to papain at the atomic level, the crystal structure of the complex was analysed by X-ray diffraction at 1.9 A (1 A is expressed in SI units as 0.1 nm) resolution. The crystal has a space group P2(1)2(1)2(1) with a = 43.37, b = 102.34 and c = 49.95 A. A total of 21,135 observed reflections were collected from the same crystal, and 14811 unique reflections of up to 1.9 A resolution [Fo > 3 sigma(Fo)] ...

  1. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  2. A Study on Solubilization of Poorly Soluble Drugs by Cyclodextrins and Micelles: Complexation and Binding Characteristics of Sulfamethoxazole and Trimethoprim

    Directory of Open Access Journals (Sweden)

    Sinem Göktürk

    2012-01-01

    > α-CD. With taking into consideration of solubilization capacity of SDS micelles, it has been found that the solubility enhancement of TMP is much higher than that of SMX in the presence of SDS micelles. The binding constants of SMX and TMP obtained from the Benesi-Hildebrand equation are also confirmed by the estimated surface properties of SDS, employing the surface tension measurements. In order to elucidate the solubilization characteristics the surface tension measurements were also performed for nonionic surfactant Triton X-100. Polarity of the microenvironment and probable location of SMX and TMP were also discussed in the presence of various organic solvents.

  3. A macrocyclic ligand as receptor and Zn(II)-complex receptor for anions in water: binding properties and crystal structures.

    Science.gov (United States)

    Ambrosi, Gianluca; Formica, Mauro; Fusi, Vieri; Giorgi, Luca; Macedi, Eleonora; Micheloni, Mauro; Paoli, Paola; Pontellini, Roberto; Rossi, Patrizia

    2011-02-01

    Binding properties of 24,29-dimethyl-6,7,15,16-tetraoxotetracyclo[19.5.5.0(5,8).0(14,17)]-1,4,9,13,18,21,24,29-octaazaenatriaconta-Δ(5,8),Δ(14,17)-diene ligand L towards Zn(II) and anions, such as the halide series and inorganic oxoanions (phosphate (Pi), sulfate, pyrophosphate (PPi), and others), were investigated in aqueous solution; in addition, the Zn(II)/L system was tested as a metal-ion-based receptor for the halide series. Ligand L is a cryptand receptor incorporating two squaramide functions in an over-structured chain that connects two opposite nitrogen atoms of the Me(2)[12]aneN(4) polyaza macrocyclic base. It binds Zn(II) to form mononuclear species in which the metal ion, coordinated by the Me(2)[12]aneN(4) moiety, lodges inside the three-dimensional cavity. Zn(II)-containing species are able to bind chloride and fluoride at the physiologically important pH value of 7.4; the anion is coordinated to the metal center but the squaramide units play the key role in stabilizing the anion through a hydrogen-bonding network; two crystal structures reported here clearly show this aspect. Free L is able to bind fluoride, chloride, bromide, sulfate, Pi, and PPi in aqueous solution. The halides are bound at acidic pH, whereas the oxoanions are bound in a wide range of pH values ranging from acidic to basic. The cryptand cavity, abundant in hydrogen-bonding sites at all pH values, allows excellent selectivity towards Pi to be achieved mainly at physiological pH 7.4. By joining amine and squaramide moieties and using this preorganized topology, it was possible, with preservation of the solubility of the receptor, to achieve a very wide pH range in which oxoanions can be bound. The good selectivity towards Pi allows its discrimination in a manner not easily obtainable with nonmetallic systems in aqueous environment. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Two Discrete RuCp* (Cp*=Pentamethylcyclopentadienyl) Binding Modes of N-Confused Porphyrins: Peripheral π Complex and Sitting Atop Ruthenocenophane Complex by Skeletal Transformation.

    Science.gov (United States)

    Yamamoto, Takaaki; Mitsuno, Koki; Mori, Shigeki; Itoyama, Shuhei; Shiota, Yoshihito; Yoshizawa, Kazunari; Ishida, Masatoshi; Furuta, Hiroyuki

    2018-05-07

    Complexation of a RuCp* cation with N-confused tetraarylporphyrins (NCPs) forms directly bound ruthenium(II) pentamethylcyclopentadienyl (Cp*) π-complex on a specific meso-aryl group (e.g., phenyl) neighboring peripheral imino nitrogen of NCPs in high yields. In contrast, in the case of NCPs bearing bulky meso-substituents (e.g., 3,5-di-tert-butylphenyl), new ruthenocenophane-like complex embedded on an N-confused calix[4]phyrin was formed through multiple C-H bond activation of methyl groups of Cp* ligand. The mechanistic insight into the formation of the ruthenocenophane was derived from DFT calculations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Binding of molecular oxygen by an artificial heme analogue: investigation on the formation of an Fe–tetracarbene superoxo complex

    KAUST Repository

    Anneser, Markus R.; Haslinger, Stefan; Pö thig, Alexander; Cokoja, Mirza; D'Elia, Valerio; Hö gerl, Manuel P.; Basset, Jean-Marie; Kü hn, Fritz E.

    2016-01-01

    The dioxygen reactivity of a cyclic iron(II) tetra–NHC-complex (NHC: N-heterocyclic carbene) is investigated. Divergent oxidation behavior is observed depending on the choice of the solvent (acetonitrile or acetone). In the first case, exposure

  6. Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding

    Science.gov (United States)

    Keren-Kaplan, Tal; Attali, Ilan; Estrin, Michael; Kuo, Lillian S; Farkash, Efrat; Jerabek-Willemsen, Moran; Blutraich, Noa; Artzi, Shay; Peri, Aviyah; Freed, Eric O; Wolfson, Haim J; Prag, Gali

    2013-01-01

    The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a Kd of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC50 of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding. PMID:23361315

  7. Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding.

    Science.gov (United States)

    Keren-Kaplan, Tal; Attali, Ilan; Estrin, Michael; Kuo, Lillian S; Farkash, Efrat; Jerabek-Willemsen, Moran; Blutraich, Noa; Artzi, Shay; Peri, Aviyah; Freed, Eric O; Wolfson, Haim J; Prag, Gali

    2013-02-20

    The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.

  8. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer.

    Science.gov (United States)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-05-01

    The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq-RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq-RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 A, while the type 2 Hfq-RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 A. Diffraction data were collected to a resolution of 2.20 A from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis.

  9. Dioxygen Binding by Cobalt(II Complexes of 8,8'-bis(aminomethyl-2,2'-biquinoline

    Directory of Open Access Journals (Sweden)

    Muhammad S. Khan

    2015-02-01

    Full Text Available A new series of Co(II complexes of the type CoLX2 , where Lis a tetra-dentate ligand 8,8'-bis(aminomethyl-2,2'-biquinoline  and X= SCN–, BF4–, I– or NO3– ,  have been synthesized and characterized. The complexes have magnetic moments in the range typical of a low-spin d7 Co(II center. The interaction of these Co(II complexes with dioxygen was studied by E.S.R. spectroscopy. A dicobalt(III peroxo-bridged complex was formed in DMF solution, associated with the formation of an E.S.R.-silent solution compatible with the formation of low-spin d6 Co(III species. Similar behavior was also observed for the interaction of these Co(II complexes with dioxygen in the presence of ancillary ligands such as pyridine and 2-methylimidazole. The dioxygen complexes proved successful as oxygen carrier oxidation catalysts for tripehnylphosphine and 2,6-bis(t-butylphenol.

  10. DNA-binding, catalytic oxidation, C—C coupling reactions and antibacterial activities of binuclear Ru(II thiosemicarbazone complexes: Synthesis and spectral characterization

    Directory of Open Access Journals (Sweden)

    Arumugam Manimaran

    2012-07-01

    Full Text Available New hexa-coordinated binuclear Ru(II thiosemicarbazone complexes of the type {[(B(EPh3(COClRu]2L} (where, E = P or As; B = PPh3 or AsPh3 or pyridine; L = mononucleating NS donor of N-substituted thiosemicarbazones have been synthesized and characterized by elemental analysis, FT-IR, UV–vis and 31P{1H} NMR cyclic voltammetric studies. The DNA-binding studies of Ru(II complexes with calf thymus DNA (CT-DNA were investigated by UV–vis, viscosity measurements, gel-electrophoresis and fluorescence spectroscopy. The new complexes have been used as catalysts in C—C coupling reaction and in the oxidation of alcohols to their corresponding carbonyl compounds by using NMO as co-oxidant and molecular oxygen (O2 atmosphere at ambient temperature. Further, the new binucleating thiosemicarbazone ligands and their Ru(II complexes were also screened for their antibacterial activity against Klebsiella pneumoniae, Shigella sp., Micrococcus luteus, Escherichia coli and Salmonella typhi. From this study, it was found out that the activity of the complexes almost reaches the effectiveness of the conventional bacteriocide.

  11. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

    Science.gov (United States)

    Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima

    2017-04-03

    The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  12. 3d/4f Metal Complexes of Phenolic Oximes New Binding Sites on Anderson Polyoxometalates Metal Complexes of the New THAME Ligand

    DEFF Research Database (Denmark)

    Sethi, Waqas

    to be coordinatedto SMMs as well as onto other transition metal complexes, via different synthetic strategies. These synthetic routes did not result in the crystallisation of any new compounds. A new hexadentate ligand tris(((2-hydroxyethyl)-amino)methyl)ethane (THAME) was synthesised and characterised. THAME...

  13. Exploring the oxidation and iron binding profile of a cyclodextrin encapsulated quercetin complex unveiled a controlled complex dissociation through a chemical stimulus.

    Science.gov (United States)

    Diamantis, Dimitrios A; Ramesova, Sarka; Chatzigiannis, Christos M; Degano, Ilaria; Gerogianni, Paraskevi S; Karadima, Constantina; Perikleous, Sonia; Rekkas, Dimitrios; Gerothanassis, Ioannis P; Galaris, Dimitrios; Mavromoustakos, Thomas; Valsami, Georgia; Sokolova, Romana; Tzakos, Andreas G

    2018-06-07

    Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used. The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry) UV-Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H 2 O 2 -induced DNA damage. Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and oxidation profile is retained in its encapsulated state. Although the protective ability of the quercetin-2HP-β-CD complex against H 2 O 2 was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin. We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity. The oxidation profile of a natural product once it is encapsulated in a supramolecular cyclodextrin carrier as also it was discovered that decomplexation can be triggered by a chemical stimulus. Copyright © 2018. Published by Elsevier B.V.

  14. 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.

    Science.gov (United States)

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

    2014-10-24

    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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. 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*

    Science.gov (United States)

    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

  16. Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Wei [Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 200032 (China); NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032 (China); Li, Juan [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025 (China); Qiu, Zhuibai [Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 200032 (China); Xia, Zheng [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025 (China); Li, Wei [Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 200032 (China); Yu, Lining; Chen, Hailin; Chen, Jianxing [NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032 (China); Chen, Yan; Hu, Zhuqin; Zhou, Wei; Shao, Biyun; Cui, Yongyao [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025 (China); Xie, Qiong, E-mail: xiejoanxq@gmail.com [Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 200032 (China); Chen, Hongzhuan, E-mail: yaoli@shsmu.edu.cn [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025 (China)

    2012-10-01

    The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC{sub 50} values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC{sub 50} values of 49.1 μM (for ZLA) and 55.3 μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.

  17. Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

    International Nuclear Information System (INIS)

    Zheng, Wei; Li, Juan; Qiu, Zhuibai; Xia, Zheng; Li, Wei; Yu, Lining; Chen, Hailin; Chen, Jianxing; Chen, Yan; Hu, Zhuqin; Zhou, Wei; Shao, Biyun; Cui, Yongyao; Xie, Qiong; Chen, Hongzhuan

    2012-01-01

    The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC 50 values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC 50 values of 49.1 μM (for ZLA) and 55.3 μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.

  18. An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

    Directory of Open Access Journals (Sweden)

    Rajendra Kumar Singh

    2009-12-01

    Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

  19. Crystallization and crystallographic analysis of the ligand-binding domain of the Pseudomonas putida chemoreceptor McpS in complex with malate and succinate

    International Nuclear Information System (INIS)

    Gavira, J. A.; Lacal, J.; Ramos, J. L.; García-Ruiz, J. M.; Krell, T.; Pineda-Molina, E.

    2012-01-01

    The crystallization of the ligand-binding domain of the methyl-accepting chemotaxis protein chemoreceptor McpS (McpS-LBD) is reported. Methyl-accepting chemotaxis proteins (MCPs) are transmembrane proteins that sense changes in environmental signals, generating a chemotactic response and regulating other cellular processes. MCPs are composed of two main domains: a ligand-binding domain (LBD) and a cytosolic signalling domain (CSD). Here, the crystallization of the LBD of the chemoreceptor McpS (McpS-LBD) is reported. McpS-LBD is responsible for sensing most of the TCA-cycle intermediates in the soil bacterium Pseudomonas putida KT2440. McpS-LBD was expressed, purified and crystallized in complex with two of its natural ligands (malate and succinate). Crystals were obtained by both the counter-diffusion and the hanging-drop vapour-diffusion techniques after pre-incubation of McpS-LBD with the ligands. The crystals were isomorphous and belonged to space group C2, with two molecules per asymmetric unit. Diffraction data were collected at the ESRF synchrotron X-ray source to resolutions of 1.8 and 1.9 Å for the malate and succinate complexes, respectively

  20. Binding of molecular oxygen by an artificial heme analogue: investigation on the formation of an Fe–tetracarbene superoxo complex

    KAUST Repository

    Anneser, Markus R.

    2016-02-26

    The dioxygen reactivity of a cyclic iron(II) tetra–NHC-complex (NHC: N-heterocyclic carbene) is investigated. Divergent oxidation behavior is observed depending on the choice of the solvent (acetonitrile or acetone). In the first case, exposure to molecular oxygen leads to an oxygen free Fe(III) whereas in the latter case an oxide bridged Fe(III) dimer is formed. In acetone, an Fe(III)-superoxide can be trapped, isolated and characterized as intermediate at low temperatures. An Fe(III)–O–Fe(III) dimer is formed from the Fe(III) superoxide in acetone upon warming and the molecular structure has been revealed by single crystal X-ray diffraction. It is shown that the oxidation of the Fe(II) complex in both solvents is a reversible process. For the regeneration of the initial Fe(II) complex both organic and inorganic reducing agents can be used.

  1. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    International Nuclear Information System (INIS)

    Paulin, Sarah; Rosado, Helena; Taylor, Peter W; Jamshad, Mohammed; Dafforn, Timothy R; Garcia-Lara, Jorge; Foster, Simon J; Galley, Nicola F; Roper, David I

    2014-01-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function. (paper)

  2. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    Science.gov (United States)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  3. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  4. Binding orientation and interaction of bile salt in its ternary complex with pancreatic lipase-colipase system.

    Science.gov (United States)

    Haque, Neshatul; Prakash Prabhu, N

    2018-05-23

    The interfacial activity of pancreatic lipases (PL) depends on the presence of colipase and bile salt. The activity of PL is inhibited by micellar concentrations of bile salt which can be restored by the addition of colipase. Though the formation of 1:1:1 tertiary complex by lipase-colipase-bile salt micelle is well accepted, the residue-level interactions between lipase-colipase and bile salt are yet to be clearly understood. Molecular dynamic simulations of lipase-colipase complex, lipase and colipase were performed in the presence of a model bile salt, sodium taurocholate (NaTC), at its near-CMC and supra-micellar concentrations. From the interactions obtained from the molecular dynamic simulations, the ternary complex was modelled and compared with earlier reports. The analysis suggested that a micelle of NaTC consisting of nine monomers was formed at the concave groove between lipase and colipase chain and it mainly interacted with the fourth finger of colipase. This complex was mainly stabilized by van der Waals interactions. Interestingly, the C-terminal domain of lipase which holds the colipase did not show any significant role in formation or stabilization of NaTC micelle. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen.

    Science.gov (United States)

    Esteves, Catarina V; Esteban-Gómez, David; Platas-Iglesias, Carlos; Tripier, Raphaël; Delgado, Rita

    2018-05-11

    The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid-base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi 3- , ATP 4- , ADP 3- , AMP 2- , PhPO 4 2- , and HPO 4 2- ). On the one hand, the acid-base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn 2 bmce(HPPi)] + . Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn 2 bmce] 4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.

  6. Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

    Science.gov (United States)

    Chen, Kuan-Yu; Li, Hsou-min

    2007-01-01

    The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

  7. Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

    Science.gov (United States)

    Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

    2011-04-08

    Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

  8. 13C and 17O NMR binding constant studies of uranyl carbonate complexes in near-neutral aqueous solution. Yucca Mountain Project Milestone Report 3351

    International Nuclear Information System (INIS)

    Clark, D.L.; Newton, T.W.; Palmer, P.D.; Zwick, B.D.

    1995-01-01

    Valuable structural information, much of it unavailable by other methods, can be obtained about complexes in solution through NMR spectroscopy. From chemical shift and intensity measurements of complexed species, NMR can serve as a species-specific structural probe for molecules in solution and can be used to validate thermodynamic constants used in geochemical modeling. Fourier-transform nuclear magnetic resonance (FT-NMR) spectroscopy has been employed to study the speciation of uranium(VI) ions in aqueous carbonate solutions as a function of pH, ionic strength, carbonate concentration, uranium concentration, and temperature. Carbon-13 and oxygen-17 NMR spectroscopy were used to monitor the fractions, and hence thermodynamic binding constants of two different uranyl species U0 2 (CO 3 ) 3 4- and (UO 2 ) 3 (CO 3 ) 6 6- in aqueous solution. Synthetic buffer solutions were prepared under the ionic strength conditions used in the NMR studies in order to obtain an accurate measure of the hydrogen ion concentration, and a discussion of pH = -log(a H + ) versus p[H] = -log[H+] is provided. It is shown that for quantitative studies, the quantity p[H] needs to be used. Fourteen uranium(VI) binding constants recommended by the OECD NEA literature review were corrected to the ionic strengths employed in the NMR study using specific ion interaction theory (SIT), and the predicted species distributions were compared with the actual species observed by multinuclear NMR. Agreement between observed and predicted stability fields is excellent. This establishes the utility of multinuclear NMR as a species-specific tool for the study of the actinide carbonate complexation constants, and serves as a means for validating the recommendations provided by the OECD NEA

  9. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    Science.gov (United States)

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-09

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

  10. Binding of chlorfenvinphos and malathion with DNA and their detection using new sensitive luminescent Tb(III) complex probe

    Energy Technology Data Exchange (ETDEWEB)

    Azab, Hassan A. [Chemistry Department, Faculty of Science, Suez Canal University, 41522 Ismailia (Egypt); Kamel, Rasha M., E-mail: rashamoka@yahoo.com [Chemistry Department, Faculty of Science, Suez University, 43518 Suez (Egypt)

    2016-02-15

    In this work, the interaction of new luminescent terbium-N(acetoacetyl)-3-allyl-2hydroxybenzaldehyde hydrazone complex in a metal ligand ratio 1:2 in ethanol was studied with different pesticides, using fluorescence technique. The results indicate that the pesticides chlorfenvinphos and malathion exhibit quenching effect on the characteristics emission peak for Tb(III) at λ=545 nm. The luminescence variations values using the probe Tb(III)(HBH){sub 2} fit Stern–Volmer equation, where the detection limits are 4.53 and 9.59 µmol/L for chlorfenvinphos and malathion, respectively. The thermodynamic parameters associated with the interaction of the complex with the two pesticides were calculated, where the reaction is spontaneous through the obtained negative values of free energy change ΔG, and the process is of exothermic nature with decrease in entropy. Hence the nature of forces acting between the complex and pesticide molecules are Vander Val's and hydrogen bond. Also, it was found that the quenching mechanism is static type. Effect of some relevant interferents on the detection of pesticides has been investigated. The new sensing complex was applied to the determination of the pesticides in different real water samples (tap, river, and waste water) with precise recovery. Also, the effect of different concentrations of pesticides on the fluorescence spectrum of DNA was studied. - Highlights: • The method can be used for detection of pesticides in environmental samples. • The DL is 4.53 and 9.59 µmol L{sup −1} for chlorfenvinphos and malathion respectively. • Stern–Volmer studies at different temperatures were studied. • Effect of some interferents on the detection of pesticides has been investigated.

  11. Intellectual disabilities, neuronal posttranscriptional RNA metabolism, and RNA-binding proteins: three actors for a complex scenario.

    Science.gov (United States)

    Bardoni, Barbara; Abekhoukh, Sabiha; Zongaro, Samantha; Melko, Mireille

    2012-01-01

    Intellectual disability (ID) is the most frequent cause of serious handicap in children and young adults and interests 2-3% of worldwide population, representing a serious problem from the medical, social, and economic points of view. The causes are very heterogeneous. Genes involved in ID have various functions altering different pathways important in neuronal function. Regulation of mRNA metabolism is particularly important in neurons for synaptic structure and function. Here, we review ID due to alteration of mRNA metabolism. Functional absence of some RNA-binding proteins--namely, FMRP, FMR2P, PQBP1, UFP3B, VCX-A--causes different forms of ID. These proteins are involved in different steps of RNA metabolism and, even if a detailed analysis of their RNA targets has been performed so far only for FMRP, it appears clear that they modulate some aspects (translation, stability, transport, and sublocalization) of a subset of RNAs coding for proteins, whose function must be relevant for neurons. Two other proteins, DYRK1A and CDKL5, involved in Down syndrome and Rett syndrome, respectively, have been shown to have an impact on splicing efficiency of specific mRNAs. Both proteins are kinases and their effect is indirect. Interestingly, both are localized in nuclear speckles, the nuclear domains where splicing factors are assembled, stocked, and recycled and influence their biogenesis and/or their organization. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Synthesis of a ruthenium(II) bipyridyl complex coordinated by a functionalized Schiff base ligand: characterization, spectroscopic and isothermal titration calorimetry measurements of M2+ binding and sensing (M2+=Ca2+, Mg2+).

    Science.gov (United States)

    Dixit, Namrata; Mishra, Lallan; Mustafi, Sourajit M; Chary, Kandala V R; Houjou, Hirohiko

    2009-07-01

    Bis-[methylsalicylidine-4'benzoic acid]-ethylene (LH2) complexed with cis-Ru(bpy)2Cl(2).2H2O provides a complex of composition [Ru(bpy)2L].2NH4PF6 (1), which has been characterized spectroscopically. Its binding behaviour towards Mg2+ and Ca2+ ions is monitored using 1H NMR titration, isothermal titration calorimetry (ITC) and luminescence microscopy. The luminescent ruthenium complex binds Ca2+ in a more selective manner as compared to Mg2+.

  13. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

    Directory of Open Access Journals (Sweden)

    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  14. Steric effects of CO2 binding to transition metal-benzene complexes: a first-principles study

    OpenAIRE

    Bae, Hyeonhu; Huang, Bing; Lee, Hoonkyung

    2016-01-01

    Using density functional theory (DFT) calculations, we investigated the adsorption of CO2 molecules on 3d transition metal (TM)-benzene complexes. Our calculations show that the maximum number of CO2 molecules adsorbable on Sc or Ti atoms is three, but the 18-electron rule predicts it should be four. The 18-electron rule is generally successful in predicting the maximum H2 adsorption number for TM atoms including Sc or Ti atoms. We found that the 18-electron rule fails to correctly predict CO...

  15. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  16. The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs.

    Science.gov (United States)

    Hecker, Andreas; Brand, Luise H; Peter, Sébastien; Simoncello, Nathalie; Kilian, Joachim; Harter, Klaus; Gaudin, Valérie; Wanke, Dierk

    2015-07-01

    Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein basic pentacysteine6 (BPC6) interacts with like heterochromatin protein1 (LHP1), a PRC1 component, and associates with vernalization2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain

    Energy Technology Data Exchange (ETDEWEB)

    Miller, D.J.; Ouellette, N.; Evodokimova, E.; Savchenko, A.; Edwards, A.; Anderson, W.F. (Toronto); (NWU)

    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.

  18. Molecular cloning and characterization of rhesus monkey platelet glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex.

    Science.gov (United States)

    Qiao, Jianlin; Shen, Yang; Shi, Meimei; Lu, Yanrong; Cheng, Jingqiu; Chen, Younan

    2014-05-01

    Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα. DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation. Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα-binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses. Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  20. Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

    Science.gov (United States)

    Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

    2017-04-15

    RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Octamer-binding protein 4 affects the cell biology and phenotypic transition of lung cancer cells involving β-catenin/E-cadherin complex degradation.

    Science.gov (United States)

    Chen, Zhong-Shu; Ling, Dong-Jin; Zhang, Yang-De; Feng, Jian-Xiong; Zhang, Xue-Yu; Shi, Tian-Sheng

    2015-03-01

    Clinical studies have reported evidence for the involvement of octamer‑binding protein 4 (Oct4) in the tumorigenicity and progression of lung cancer; however, the role of Oct4 in lung cancer cell biology in vitro and its mechanism of action remain to be elucidated. Mortality among lung cancer patients is more frequently due to metastasis rather than their primary tumors. Epithelial‑mesenchymal transition (EMT) is a prominent biological event for the induction of epithelial cancer metastasis. The aim of the present study was to investigate whether Oct4 had the capacity to induce lung cancer cell metastasis via the promoting the EMT in vitro. Moreover, the effect of Oct4 on the β‑catenin/E‑cadherin complex, associated with EMT, was examined using immunofluorescence and immunoprecipitation assays as well as western blot analysis. The results demonstrated that Oct4 enhanced cell invasion and adhesion accompanied by the downregulation of epithelial marker cytokeratin, and upregulation of the mesenchymal markers vimentin and N‑cadherin. Furthermore, Oct4 induced EMT of lung cancer cells by promoting β‑catenin/E‑cadherin complex degradation and regulating nuclear localization of β‑catenin. In conclusion, the present study indicated that Oct4 affected the cell biology of lung cancer cells in vitro through promoting lung cancer cell metastasis via EMT; in addition, the results suggested that the association and degradation of the β‑catenin/E‑cadherin complex was regulated by Oct4 during the process of EMT.

  2. Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome.

    Science.gov (United States)

    Pech, Markus; Spreter, Thomas; Beckmann, Roland; Beatrix, Birgitta

    2010-06-18

    Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of betaNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of betaNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, alphaNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.

  3. Direction of Intercalation of a bis-Ru(II) Complex to DNA Probed by a Minor Groove Binding Molecule 4',6-Diamidino-2-phenylindole

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Yoon Jung; Kim, Raeyeong; Chitrapriya, Nataraj; Kim, Seog K.; Bae, Inho [Yeungnam Univ., Gyeongsan (Korea, Republic of)

    2013-10-15

    Direction of intercalation to DNA of the planar dipyrido[3,2-a:2',3'-c]phenazine ligands (dppz) of a bis-Ru(II) complex namely, [Ru(1,10-phenanthroline){sub 2}dipyrido[3,2-a:2',3'-c]phenazine]{sup 2+} linkered by a 1,3-bis(4-pyridyl)propane, was investigated by probing the behavior of 4',6-diamidino-2-phenylindole (DAPI) that bound deep in the minor groove. Bis-intercalation of DPPZ resulted in a little blue shift and hyperchromism in DAPI absorption band, and a large decrease in DAPI fluorescence intensity which accompanied by an increase in the dppz emission intensity. Diminishing the intensity of the positive induced circular dichroism (CD) and linear dichroism (LD) were also observed. These spectral changes indicated that insertion of dppz ligand caused the change of the binding mode of DAPI, which probably moved to the exterior of DNA from the minor groove and interacted with the phospghate groups of DNA by electrostatic interaction. At the surface of DNA, DAPI binds at the phosphate groups of DNA by electrostatic attraction. Consequently, this observation indicated that the dppz ligand intercalated from the minor groove.

  4. Neutron scattering with deuterium labeling reveals the nature of complexes formed by Ca{sup 2+}-binding proteins and their regulatory targets

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Small-angle neutron scattering with deuterium labeling is extremely useful for studying the structures of complex biomolecular assemblies in solution. The different neutron scattering properties of their isotopes of hydrogen combines with the ability to uniformly label biomolecules with deuterium allow one to characterize the structures and relative dispositions of the individual components of an assembly using methods of {open_quotes}contrast variation.{close_quotes} We have applied these techniques to studies of the evolutionarily related dumbbell-shaped Ca{sup 2+}-binding proteins calmodulin and troponin C and their interactions with the target proteins whose activities they regulate. Ca{sup 2+} is one of the simplest of nature`s messengers used in the communication pathways between physiological stimulus and cellular response. The signaling mechanism generally involves Ca{sup 2+} binding to a protein and inducing a conformational change that transmits a signal to modify the activity of a specific target protein. Ca{sup 2+} is thus important in the regulation of a diverse array of intracellular responses, including neurotransmitter release, muscle contraction, the degradation of glycogen to glucose to generate energy, microtubule assembly, membrane phosphorylation, etc. It is the conformational language of the Ca{sup 2+} induced signal transduction that we have sought to understand because of its central importance to biochemical regulation and, hence, to healthy cellular function.

  5. Ring-to-chain conformation may be a determining factor in the ability of xanthophylls to bind to the bulk light-harvesting complex of plants

    Science.gov (United States)

    Young, Andrew J.; Phillip, Denise M.; Hashimoto, Hideki

    2002-12-01

    The binding of xanthophylls to the main light-harvesting complex (LHC) of higher plants has been studied using the technique of in vitro reconstitution. This demonstrated that the carotenoid diol lactucaxanthin (native to many LHC) would not support the assembly of LHC whilst other diols, notably zeaxanthin and lutein would. Analysis of the most stable forms of the carotenoid end-groups found in xanthophylls native to higher plant LHC (as determined by theoretical calculations) revealed profound differences in the adiabatic potential energy curves for the C5-C6-C7-C8-torsion angle for the ɛ end-groups in lactucaxanthin (6-s- trans), in comparison to carotenoids possessing a 3-hydroxy β end-group (zeaxanthin; 6-s- cis), 3-hydroxy-4-keto β end-group (astaxanthin, 6-s- cis) or a 3-hydroxy-5,6-epoxy end-group (violaxanthin, distorted 6-s- cis). The (ɛ end-groups of other carotenoids studied were 6-s- trans. We examine the possible relationship between carotenoid ring-to-chain conformation and binding to LHC.

  6. Synthesis and characterization of nitrile functionalized silver(I)-N-heterocyclic carbene complexes: DNA binding, cleavage studies, antibacterial properties and mosquitocidal activity against the dengue vector, Aedes albopictus.

    Science.gov (United States)

    Asekunowo, Patrick O; Haque, Rosenani A; Razali, Mohd R; Avicor, Silas W; Wajidi, Mustafa F F

    2018-04-25

    A series of four benzimidazolium based nitrile-functionalized mononuclear-Ag(I)-N-heterocyclic carbene and binuclear-Ag(I)-N-heterocyclic carbene (Ag(I)-NHC) hexafluorophosphate complexes (5b-8b) were synthesized by reacting the corresponding hexafluorophosphate salts (1b-4b) with Ag 2 O in acetonitrile, respectively. These compounds were characterized by 1 H NMR, 13 C NMR, IR, UV-visible spectroscopic techniques, elemental analyses and molar conductivity. Additionally, 8b was structurally characterized by single crystal X-ray diffraction technique. Preliminary in vitro antibacterial evaluation was conducted for all the compounds against two standard bacteria; gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacterial strains. Most of the Ag(I)-NHC complexes (5b-8b) showed moderate to good antibacterial activity with MIC values in the range of 12.5-100 μg/mL. Especially, compound 8b exhibited promising anti-Staphylococcus aureus activity with a low MIC value (12.5 μg/mL). However, all the hexafluorophosphate salts (1b-4b) were inactive against the bacteria strains. The preliminary interactive investigation revealed that the most active compound, 8b, could effectively intercalate into DNA to form 8b-DNA complex which shows a better binding ability for DNA (K b  = 3.627 × 10 6 ) than the complexes 5b-7b (2.177 × 10 6 , 8.672 × 10 5 and 6.665 × 10 5 , respectively). Nuclease activity of the complexes on plasmid DNA and Aedes albopictus genomic DNA was time-dependent, although minimal. The complexes were larvicidal to the mosquito, with 5b, 6b and 8b being highly active. Developmental progression from the larval to the adult stage was affected by the complexes, progressively being toxic to the insect's development with increasing concentration. These indicate the potential use of these complexes as control agents against bacteria and the dengue mosquito Ae. albopictus. Copyright © 2018 Elsevier Masson SAS. All

  7. Development and clinical evaluation of a novel immunoassay for the binary complex of IGF-I and IGF-binding protein-1 in human serum

    DEFF Research Database (Denmark)

    Frystyk, Jan; Højlund, Kurt; Rasmussen, Kirsten Nyborg

    2002-01-01

    Correlation studies have suggested that IGF-binding protein (IGFBP)-1 is a dynamic regulator of free IGF-I. To further study this, we developed a monoclonal immunofluorometric assay specific for the binary complex of IGF-I and IGFBP-1 in human serum. An IGFBP-1 antibody, which recognizes all...... phospho-forms of IGFBP-1, was used for coating. An europium-labeled IGF-I antibody served as tracer. Assay incubation was performed at conditions approaching those in vivo (i.e. pH 7.4, 37 C). The assay was highly specific: no signal was obtained unless both IGF-I and IGFBP-1 were present and neither...

  8. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    Science.gov (United States)

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

  9. Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5α-androst-16-en-3α-ol

    International Nuclear Information System (INIS)

    Vincent, Jeremy; Shan, Li; Fan, Ming; Brunzelle, Joseph S.; Forman, Barry M.; Fernandez, Elias J.

    2004-01-01

    The purification and structure determination of the murine constitutive androstane receptor bound to its inverse agonist/antagonist androstenol is described. The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement

  10. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: tseo@dongguk.edu [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  11. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    International Nuclear Information System (INIS)

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-01-01

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  12. Diverse CdII coordination complexes derived from bromide isophthalic acid binding with auxiliary N-donor ligands

    International Nuclear Information System (INIS)

    Tang, Meng; Dong, Bao-Xia; Wu, Yi-Chen; Yang, Fang; Liu, Wen-Long; Teng, Yun-Lei

    2016-01-01

    The coordination characteristics of 4-bromoisophthalic acid (4-Br-H 2 ip) have been investigated in a series of Cd II -based frameworks. Hydrothermal reactions of Cd II salts and 4-Br-H 2 ip together with flexible or semiflexible N-donor auxiliary ligands resulted in the formation of four three-dimensional coordination complexes with diverse structures: (Cd(bix) 0.5 (bix) 0.5 (4-Br-ip)]·H 2 O) n (1), [Cd(bbi) 0.5 (bbi) 0.5 (4-Br-ip)] n (2), ([Cd(btx) 0.5 (4-Br-ip)(H 2 O)]·0.5CH 3 OH·H 2 O) n (3) and ([Cd(bbt) 0.5 (4-Br-ip)(H 2 O)]·3·5H 2 O) n (4). These compounds were characterized by elemental analyses, IR spectra, single-crystal and powder X-ray diffraction. They displayed diverse structures depending on the configuration of the 4-connected metal node, the coordination mode of the 4-Br-H 2 ip, the coordination ability and conformationally flexibility of the N-donor auxiliary. Compound 1 exhibits 3-fold interpenetrated 6 6 topology and compound 2 has a 4 12 topology. Compounds 3–4 have similar 3D pillar-layered structures based on 3,4-connected binodal net with the Schläfli symbol of (4·3 8 ). The thermal stabilities and photoluminescence properties of them were discussed in detail. - Graphical abstract: Four 3D Cd II coordination complexes on the basis of 4-bromoisophthalic acid (4-Br-H 2 ip) and two types of flexible (bbi, bbt) and semiflexible (bix, btx) N-donor ligands are prepared. They displayed diverse topology structures of 6 6 (1), 4 12 (2) and 4·3 8 (3−4), depending on the configuration of the 4-connected metal node, the coordination mode of the 4-Br-H 2 ip, the coordination ability and conformationally flexibility of the N-donor auxiliary ligand. - Highlights: • Four 3D Cd II coordination complexes based on 4-Br-H 2 ip and flexible/semiflexible N-donor ligands have been synthesized. • They displayed diverse topology structures of 6 6 for 1, 4 12 for 2 and 4·3 8 for 3–4. • The structural diversity depends on the configuration of 4-Br

  13. The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group.

    Science.gov (United States)

    Phillip, Denise; Hobe, Stephan; Paulsen, Harald; Molnar, Peter; Hashimoto, Hideki; Young, Andrew J

    2002-07-12

    The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.

  14. Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR

    Directory of Open Access Journals (Sweden)

    Esbelin Julia

    2012-06-01

    Full Text Available Abstract Background Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr. Results Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]2+ cluster per monomer. Atmospheric O2 causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex. Conclusions Overall, this work shows that incorporation of the [4Fe-4S]2+ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

  15. Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis

    DEFF Research Database (Denmark)

    Sedgwick, G.G.; Townsend, K.; Martin, A.

    2013-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC....../C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates......, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition...

  16. An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

    Science.gov (United States)

    Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

    2017-11-08

    The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

  17. The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication.

    Science.gov (United States)

    Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

    2013-03-08

    The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1(+), two temperature-sensitive mcb1 gene mutants (mcb1(ts)) were isolated. Extensive genetic analysis showed that the mcb1(ts) mutants were suppressed by a mcm5(+) multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1(ts) mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1(ts) mutants. Furthermore, the mcb1(ts) mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.

  18. The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication*

    Science.gov (United States)

    Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

    2013-01-01

    The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1+, two temperature-sensitive mcb1 gene mutants (mcb1ts) were isolated. Extensive genetic analysis showed that the mcb1ts mutants were suppressed by a mcm5+ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1ts mutants. Furthermore, the mcb1ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. PMID:23322785

  19. Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

    International Nuclear Information System (INIS)

    Yoga, Yano M. K.; Traore, Daouda A. K.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

    2011-01-01

    The successful preparation of a mutant KH domain representing the first KH domain of PCBP1 and its crystallization in complex with a C-rich DNA are reported. This structure is anticipated to provide high-resolution information that will allow better understanding of the basis of cytosine specificity by PCBPs. Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1–DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate–citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2 1 , with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1–DNA complex will further our insight into the basis of cytosine specificity by PCBPs

  20. Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

    Science.gov (United States)

    Al Abo, Muthana; Dejsuphong, Donniphat; Hirota, Kouji; Yonetani, Yasukazu; Yamazoe, Mitsuyoshi; Kurumizaka, Hitoshi; Takeda, Shunichi

    2014-02-01

    BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.

  1. Drastic Effect of the Peptide Sequence on the Copper-Binding Properties of Tripeptides and the Electrochemical Behaviour of Their Copper(II) Complexes.

    Science.gov (United States)

    Mena, Silvia; Mirats, Andrea; Caballero, Ana B; Guirado, Gonzalo; Barrios, Leoní A; Teat, Simon J; Rodriguez-Santiago, Luis; Sodupe, Mariona; Gamez, Patrick

    2018-04-06

    The binding and electrochemical properties of the complexes Cu II -HAH, Cu II -HWH, Cu II -Ac-HWH, Cu II -HHW, and Cu II -WHH have been studied by using NMR and UV/Vis spectroscopies, CV, and density functional calculations. The results obtained highlight the importance of the peptidic sequence on the coordination properties and, consequently, on the redox properties of their Cu II complexes. For Cu II -HAH and Cu II -HWH, no cathodic processes are observed up to -1.2 V; that is, the complexes exhibit very high stability towards copper reduction. This behaviour is associated with the formation of very stable square-planar (5,5,6)-membered chelate rings (ATCUN motif), which enclose two deprotonated amides. In contrast, for non-ATCUN Cu II -Ac-HWH, Cu II -HHW complexes, simulations seem to indicate that only one deprotonated amide is enclosed in the coordination sphere. In these cases, the main electrochemical feature is a reductive irreversible one electron-transfer process from Cu II to Cu I , accompanied with structural changes of the metal coordination sphere and reprotonation of the amide. Finally, for Cu II -WHH, two major species have been detected: one at low pH (10) with an ATCUN motif, both species coexisting at intermediate pH. The present study shows that the use of CV, using glassy carbon as a working electrode, is an ideal and rapid tool for the determination of the redox properties of Cu II metallopeptides. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

    Science.gov (United States)

    Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

    2015-01-01

    Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

  3. Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

    International Nuclear Information System (INIS)

    Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko

    2008-01-01

    The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V M value of 3.56 Å 3 Da −1 . Diffraction data were collected to a resolution of 3.0 Å

  4. Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

    Energy Technology Data Exchange (ETDEWEB)

    Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

    2008-03-01

    The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V{sub M} value of 3.56 Å{sup 3} Da{sup −1}. Diffraction data were collected to a resolution of 3.0 Å.

  5. DNA binding, cytotoxicity and apoptosis induction activity of a mixed-ligand copper(II) complex with taurine Schiff base and imidazole

    Science.gov (United States)

    Li, Mei; kong, Lin Lin; Gou, Yi; Yang, Feng; Liang, Hong

    2014-07-01

    A novel binuclear copper(II) complex (complex 1) with taurine Schiff base and imidazole has been synthesized and structurally characterized by single crystal X-ray diffraction, elemental analysis, ESI-MS spectrometry, UV-vis and IR spectroscopy. Single-crystal analysis revealed that 1 displays the sulfonate-bridged dinuclear copper(II) centers. Both copper atoms are five-coordinated and exhibit slightly distorted square pyramidal geometries. Each of copper atom is surrounded by three oxygen atoms and one nitrogen atom from different taurine Schiff base ligands, and one nitrogen atom from one imidazole ligand. The interaction between 1 and calf thymus DNA (CT-DNA) was investigated by UV-vis, fluorescence, circular dichroism (CD) spectra and agarose gel electrophoresis. The experimental results indicated that 1 could bind to CT-DNA via an intercalative mode and show efficient cleavage activity. In addition, 1 showed an antitumor effect on cell cycle and apoptosis. Flow cytometric analysis revealed that MGC-803 cells were arrested in the S phase after treatment with 1. Fluorescence microscopic observation indicated that 1 could induce apoptosis of MGC-803 cells.

  6. Development and clinical evaluation of a novel immunoassay for the binary complex of IGF-I and IGF-binding protein-1 in human serum

    DEFF Research Database (Denmark)

    Frystyk, Jan; Højlund, Kurt; Rasmussen, Kirsten Nyborg

    2002-01-01

    Correlation studies have suggested that IGF-binding protein (IGFBP)-1 is a dynamic regulator of free IGF-I. To further study this, we developed a monoclonal immunofluorometric assay specific for the binary complex of IGF-I and IGFBP-1 in human serum. An IGFBP-1 antibody, which recognizes all...... phospho-forms of IGFBP-1, was used for coating. An europium-labeled IGF-I antibody served as tracer. Assay incubation was performed at conditions approaching those in vivo (i.e. pH 7.4, 37 C). The assay was highly specific: no signal was obtained unless both IGF-I and IGFBP-1 were present and neither...... IGFBP-2, -3, -4, nor IGF-II caused any cross-reaction. The linear standard curve covered 3 orders of magnitude, and within and in-between assay coefficients of variation were less than 5 and 15%, respectively. To study the dynamic relationship between free IGF-I and binary complex formation, seven...

  7. Robust binding between carbon nitride nanosheets and a binuclear ruthenium(II) complex enabling durable, selective CO{sub 2} reduction under visible light in aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Kuriki, Ryo; Ishitani, Osamu; Maeda, Kazuhiko [Department of Chemistry, School of Science, Tokyo Institute of Technology (Japan); Yamamoto, Muneaki; Yoshida, Tomoko [Advanced Research Institute for Natural Science and Technology, Osaka City University (Japan); Higuchi, Kimitaka; Yamamoto, Yuta; Akatsuka, Masato; Yagi, Shinya [Institute of Materials and Systems for Sustainability, Nagoya University (Japan); Lu, Daling [Suzukakedai Materials Analysis Division, Technical Department, Tokyo Institute of Technology, Yokohama (Japan)

    2017-04-18

    Carbon nitride nanosheets (NS-C{sub 3}N{sub 4}) were found to undergo robust binding with a binuclear ruthenium(II) complex (RuRu') even in basic aqueous solution. A hybrid material consisting of NS-C{sub 3}N{sub 4} (further modified with nanoparticulate Ag) and RuRu' promoted the photocatalytic reduction of CO{sub 2} to formate in aqueous media, in conjunction with high selectivity (approximately 98 %) and a good turnover number (>2000 with respect to the loaded Ru complex). These represent the highest values yet reported for a powder-based photocatalytic system during CO{sub 2} reduction under visible light in an aqueous environment. We also assessed the desorption of RuRu' from the Ag/C{sub 3}N{sub 4} surface, a factor that can contribute to a loss of activity. It was determined that desorption is not induced by salt additives, pH changes, or photoirradiation, which partly explains the high photocatalytic performance of this material. (copyright 2017 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  8. Synthesis and structural characterization of ternary Cu (II) complexes of glycine with 2,2'-bipyridine and 2,2'-dipyridylamine. The DNA-binding studies and biological activity.

    Science.gov (United States)

    Mohamed, Mervat S; Shoukry, Azza A; Ali, Ayat G

    2012-02-01

    In this study two new complexes [Cu(bpy)(Gly)Cl]·2H(2)O (1) and [Cu(dpa)(Gly)Cl]·2H(2)O (2) (bpy=2,2'-bipyridine; dpa=2,2'-dipyridylamine, Gly=glycine) have been synthesized and characterized by elemental analysis, IR, TGA, UV-vis and magnetic susceptibility measurements. The binding properties of the complexes with CT-DNA were investigated by electronic absorption spectra. The intrinsic binding constants (K(b)) calculated from UV-vis absorption studies were 1.84 × 10(3) M(-1) and 3.1 × 10(3) M(-1) for complexes 1 and 2 respectively. Thermal denaturation has been systematically studied by spectrophotometric method and the calculated ΔT(m) was nearly 5 °C for each complex. All the results suggest that the interaction modes between the complexes and CT-DNA were electrostatic and/or groove binding. The redox behavior of the two complexes was investigated by cyclic voltammetry. Both complexes, in presence and absence of CT-DNA show a quasi-reversible wave corresponding to Cu(II)/Cu(I) redox couple. The change in E(1/2), ΔE and I(pc)/I(pa) ascertain the interaction of complexes 1 and 2 with CT-DNA. Further insight into the binding of complexes with CT-DNA has been made by gel electrophoresis, where the binding of complexes is confirmed through decreasing the mobility and intensity of DNA bands. In addition, the antitumor activity of the complexes was tested on two cancer cell lines; the breast cancer (MCF7) and the human hepatocellular carcinoma (HEPG2), as well as one normal cell line; the human normal melanocytes (HFB4). The results showed that complex 1 was more potent antitumor agent than complex 2. The in-vitro antimicrobial activity of the two complexes was carried out using the disc diffusion method against different species of pathogenic bacteria and fungi. The activity data showed that complex 2 was more active in inhibiting the growth of the tested organisms. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Homology modeling and docking of AahII-Nanobody complexes reveal the epitope binding site on AahII scorpion toxin.

    Science.gov (United States)

    Ksouri, Ayoub; Ghedira, Kais; Ben Abderrazek, Rahma; Shankar, B A Gowri; Benkahla, Alia; Bishop, Ozlem Tastan; Bouhaouala-Zahar, Balkiss

    2018-02-19

    Scorpion envenoming and its treatment is a public health problem in many parts of the world due to highly toxic venom polypeptides diffusing rapidly within the body of severely envenomed victims. Recently, 38 AahII-specific Nanobody sequences (Nbs) were retrieved from which the performance of NbAahII10 nanobody candidate, to neutralize the most poisonous venom compound namely AahII acting on sodium channels, was established. Herein, structural computational approach is conducted to elucidate the Nb-AahII interactions that support the biological characteristics, using Nb multiple sequence alignment (MSA) followed by modeling and molecular docking investigations (RosettaAntibody, ZDOCK software tools). Sequence and structural analysis showed two dissimilar residues of NbAahII10 CDR1 (Tyr27 and Tyr29) and an inserted polar residue Ser30 that appear to play an important role. Indeed, CDR3 region of NbAahII10 is characterized by a specific Met104 and two negatively charged residues Asp115 and Asp117. Complex dockings reveal that NbAahII17 and NbAahII38 share one common binding site on the surface of the AahII toxin divergent from the NbAahII10 one's. At least, a couple of NbAahII10 - AahII residue interactions (Gln38 - Asn44 and Arg62, His64, respectively) are mainly involved in the toxic AahII binding site. Altogether, this study gives valuable insights in the design and development of next generation of antivenom. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  11. Comparative In Vitro Binding Studies of TiCl2(dpme2, Ti(ada2(bzac2, and TiCl2(bzac(bpme Titanium Complexes with Calf-Thymus DNA

    Directory of Open Access Journals (Sweden)

    Pamita Awasthi

    2015-01-01

    Full Text Available The binding of TiCl2(dpme2 (1, (dpme = 6,6′-dimethyl-2,2′-bipyridine, Ti(ada2(bzac2 (2, (ada = adamantylamine; bzac = benzoylacetone, and TiCl2(bzac(bpme (3, (bpme = 4,4′-dimethyl-2,2′-bipyrdine with calf thymus (ct DNA has been studied by UV-visible spectroscopy, thermal denaturation, and circular dichroism spectroscopy. In UV-visible study complexes 1, 2, and 3 showed red, blue, and red shifts, respectively, upon the addition of ct-DNA along with a significant hyperchromism. The intrinsic binding constants (Kb calculated from UV-visible absorption studies were 2.3 × 103 M−1, 3.3 × 103 M−1 and, 7.1 × 103 M−1 for complexes 1, 2, and 3, respectively. The change in melting temperature (ΔTm was calculated to be 2-3°C for each complex. Circular dichroism (CD study showed blue shift for complex 2 and red shift for complexes 1 and 3 along with rise in molecular ellipticity upon the addition of complexes. Results suggest a binding mode of complex 2 different than 1 and 3.

  12. New palladium(II) and platinum(II) 5,5-diethylbarbiturate complexes with 2-phenylpyridine, 2,2'-bipyridine and 2,2'-dipyridylamine: synthesis, structures, DNA binding, molecular docking, cellular uptake, antioxidant activity and cytotoxicity.

    Science.gov (United States)

    Icsel, Ceyda; Yilmaz, Veysel T; Kaya, Yunus; Samli, Hale; Harrison, William T A; Buyukgungor, Orhan

    2015-04-21

    Novel palladium(ii) and platinum(ii) complexes of 5,5-diethylbarbiturate (barb) with 2-phenylpyridine (Hppy), 2,2'-bipyridine (bpy) and 2,2'-dipyridylamine (dpya) have been prepared and characterized by elemental analysis, IR, UV-Vis, NMR and ESI-MS. Single-crystal diffraction measurements show that complex consists of binuclear [Pd2(μ-barb-κN,O)2(ppy-κN,C)2] moieties, while complexes are mononuclear, [M(barb-κN)2(L-κN,N')] (L = bpy or dpya). has a composition of [Pt(dpya-κN,N')2][Ag(barb-κN)2]2·4H2O and was assumed to have a structure of [Pt(barb-κN)(Hppy-κN)(ppy-κN,C)]·3H2O. The complexes were found to exhibit significant DNA binding affinity by a non-covalent binding mode, in accordance with molecular docking studies. In addition, complexes and displayed strong binding with supercoiled pUC19 plasmid DNA. Cellular uptake studies were performed to assess the subcellular localization of the selected complexes. A moderate radical scavenging activity of and was confirmed by DPPH and ABTS tests. Complexes , , and showed selectivity against HT-29 (colon) cell line.

  13. Investigation of the complex structure, comparative DNA-binding and DNA cleavage of two water-soluble mono-nuclear lanthanum(III) complexes and cytotoxic activity of chitosan-coated magnetic nanoparticles as drug delivery for the complexes

    Czech Academy of Sciences Publication Activity Database

    Asadi, Z.; Nasrollahi, N.; Karbalaei-Heidari, H.; Eigner, Václav; Dušek, Michal; Mobaraki, N.; Pournejati, R.

    2017-01-01

    Roč. 178, May (2017), s. 125-135 ISSN 1386-1425 R&D Projects: GA ČR(CZ) GA15-12653S; GA MŠk LO1603 EU Projects: European Commission(XE) CZ.2.16/3.1.00/24510 Institutional support: RVO:68378271 Keywords : lanthanum(III) * binding constant * molecular docking * DNA cleavage * cytotoxicity * chitosan Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 2.536, year: 2016

  14. 99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding

    International Nuclear Information System (INIS)

    Ono, Masahiro; Arano, Yasushi; Mukai, Takahiro; Fujioka, Yasushi; Ogawa, Kazuma; Uehara, Tomoya; Saga, Tsuneo; Konishi, Junji; Saji, Hideo

    2001-01-01

    6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ( 99m Tc)-labeled polypeptides with tricine as a coligand. However, 99m Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [ 99m Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare 99m Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [ 99m Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [ 99m Tc](HYNIC-Fab)(tricine) 2. When injected into mice, [ 99m Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [ 99m Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [ 99m Tc](HYNIC-NGA)(tricine) 2. In addition, [ 99m Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [ 99m Tc](HYNIC-NGA)(tricine) 2. Similar results were observed with 99m Tc-labeled Fab fragments where [ 99m Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [ 99m Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [ 99m Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [ 99m Tc](HYNIC)(tricine)(BP) complex

  15. Spectroscopic investigations on the complexation of Cm(III) and Eu(III) with organic model ligands and their binding mode in human urine (in vitro)

    International Nuclear Information System (INIS)

    Heller, Anne

    2011-01-01

    In case of incorporation, trivalent actinides (An(III)) and lanthanides (Ln(III)) pose a serious health risk to humans. An(III) are artificial, highly radioactive elements which are mainly produced during the nuclear fuel cycle in nuclear power plants. Via hazardous accidents or nonprofessional storage of radioactive waste, they can be released in the environment and enter the human food chain. In contrast, Ln(III) are nonradioactive, naturally occurring elements with multiple applications in technique and medicine. Consequently it is possible that humans get in contact and incorporate both, An(III) and Ln(III). Therefore, it is of particular importance to elucidate the behaviour of these elements in the human body. While macroscopic processes such as distribution, accumulation and excretion are studied quite well, knowledge about the chemical binding form (speciation) of An(III) and Ln(III) in various body fluids is still sparse. In the present work, for the first time, the speciation of Cm(III) and Eu(III) in natural human urine (in vitro) has been investigated spectroscopically and the formed complex identified. For this purpose, also basic investigations on the complex formation of Cm(III) and Eu(III) in synthetic model urine as well as with the urinary relevant, organic model ligands urea, alanine, phenylalanine, threonine and citrate have been performed and the previously unknown complex stability constants determined. Finally, all experimental results were compared to literature data and predictions calculated by thermodynamic modelling. Since both, Cm(III) and Eu(III), exhibit unique luminescence properties, particularly the suitability of time-resolved laser-induced fluorescence spectroscopy (TRLFS) could be demonstrated as a method to investigate these metal ions in untreated, complex biofluids. The results of this work provide new scientific findings on the biochemical reactions of An(III) and Ln(III) in human body fluids on a molecular scale and

  16. Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein

    Science.gov (United States)

    Satheshkumar, Angupillai; Elango, Kuppanagounder P.

    2014-09-01

    The spectral techniques such as UV-Vis, 1H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3 × 108 L mol-1. Based on the Forster’s theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol-1, which is comparable to our experimental free energy of binding (-49 kJ mol-1) obtained from fluorescence study.

  17. CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

    Science.gov (United States)

    Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

    2017-01-01

    CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

  18. Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

    DEFF Research Database (Denmark)

    Fismen, S; Hedberg, A; Fenton, K A

    2009-01-01

    Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal i......Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo......-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic...... (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i...

  19. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

    Science.gov (United States)

    Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E

    2015-08-01

    The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

  20. Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB

    Czech Academy of Sciences Publication Activity Database

    Trtková, K.; Pašková, L.; Matijescuková, N.; Strnad, Miroslav

    2010-01-01

    Roč. 57, č. 5 (2010), s. 406-414 ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50380511 Keywords : Binding * SMRT/N-CoR * complex Subject RIV: FD - Oncology ; Hematology Impact factor: 1.449, year: 2010

  1. Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

    Science.gov (United States)

    Lu, Defen; Shang, Guijun; Zhang, Heqiao; Yu, Qian; Cong, Xiaoyan; Yuan, Jupeng; He, Fengjuan; Zhu, Chunyuan; Zhao, Yanyu; Yin, Kun; Chen, Yuanyuan; Hu, Junqiang; Zhang, Xiaodan; Yuan, Zenglin; Xu, Sujuan; Hu, Wei; Cang, Huaixing; Gu, Lichuan

    2014-06-01

    The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex. © 2014 John Wiley & Sons Ltd.

  2. Analysis of some optical properties of a native and reconstituted photosystem II antenna complex, CP29 : Pigment binding sites can be occupied by chlorophyll a or chlorophyll b and determine spectral forms

    NARCIS (Netherlands)

    Giuffra, Elisabetta; Zucchelli, Giuseppe; Sandona, Dorianna; Croce, Roberta; Cugini, Daniela; Garlaschi, Flavio M.; Bassi, Roberto; Jennings, Robert C.

    1997-01-01

    The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b

  3. The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

    Science.gov (United States)

    Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

    2018-01-07

    The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

  4. Structure of a SUMO-binding-motif mimic bound to Smt3p–Ubc9p: conservation of a noncovalent Ubiquitin-like protein–E2 complex as a platform for selective interactions within a SUMO pathway

    Science.gov (United States)

    Duda, David M.; van Waardenburg, Robert C. A. M.; Borg, Laura A.; McGarity, Sierra; Nourse, Amanda; Waddell, M. Brett; Bjornsti, Mary-Ann; Schulman, Brenda A.

    2007-01-01

    Summary The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast S. cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 Å resolution crystal structure of a noncovalent Smt3p–Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the noncovalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds noncovalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the noncovalent Smt3p–Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p–Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p–Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that noncovalent ubiquitin-like protein–E2 complexes are conserved platforms, which function as parts of larger assemblies involved many protein post-translational regulatory pathways. PMID:17475278

  5. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    International Nuclear Information System (INIS)

    Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi; Hiratake, Jun; Wada, Kei

    2014-01-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp 2 hybridization to Thr403 O γ , the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs

  6. Crystal structure, DNA binding, cleavage, antioxidant and antibacterial studies of Cu(II), Ni(II) and Co(III) complexes with 2-((furan-2-yl)methylimino)methyl)-6-ethoxyphenol Schiff base

    Science.gov (United States)

    Venkateswarlu, Kadtala; Kumar, Marri Pradeep; Rambabu, Aveli; Vamsikrishna, Narendrula; Daravath, Sreenu; Rangan, Krishnan; Shivaraj

    2018-05-01

    Three novel binary metal complexes; 1 [Cu(L)2], 2 [Ni(L)2] and 3 [Co(L)3] where, L (2-(((furan-2-yl) methylimino)methyl)-6-ethoxyphenol, C14H15NO3), were synthesized and characterized by various spectral techniques. Based on spectral studies square planar geometry is assigned for Cu(II) and Ni(II) complexes, whereas Co(III) owned octahedral geometry. Ligand, [Cu(L)2] and [Ni(L)2] are crystallized and found to be monoclinic crystal systems. CT-DNA absorption binding studies revealed that the complexes show good binding propensity (Kb = 5.02 × 103 M-1, 2.77 × 103 M-1, 1.63 × 104 M-1 for 1, 2 and 3 respectively). The role of these complexes in the oxidative and photolytic cleavage of supercoiled pBR322 DNA was studied and found that the complexes cleave the pBR322 DNA effectively. The catalytic ability of 1, 2 and 3 follows the order: 3 > 1 >2. Antioxidant studies of the new complexes revealed that they exhibit significant antioxidant activity against DPPH radical. The Schiff base and its metal complexes have been screened for antibacterial studies by Minimum Inhibitory Concentration method. It is observed that all metal complexes showed more activity than free ligand.

  7. Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced rhodobacter capsulatus cytochrome c(2) to the cytochrome bc(1) complex mediated by the conformation of the rieske iron-sulfur protein

    International Nuclear Information System (INIS)

    Devanathan, S.; Salamon, Z.; Tollin, G.; Fitch, J.C.; Meyer, T.E.; Berry, E.A.; Cusanovich, M.A.

    2007-01-01

    The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to the oxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 M. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 binds less strongly (Kd = 0.11 M) but ∼30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c

  8. Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

    NARCIS (Netherlands)

    Tsuprun, V.L.; Boekema, E.J.; Samsonidze, T.G.; Pushkin, A.V.

    1991-01-01

    The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of

  9. The structure of a mixed GluR2 ligand-binding core dimer in complex with (S)-glutamate and the antagonist (S)-NS1209

    DEFF Research Database (Denmark)

    Kasper, Christina; Pickering, Darryl S; Mirza, Osman

    2006-01-01

    domains has been observed. (S)-NS1209 adopts a novel binding mode, including hydrogen bonding to Tyr450 and Gly451 of D1. Parts of (S)-NS1209 occupy new areas of the GluR2 ligand-binding cleft, and bind near residues that are not conserved among receptor subtypes. The affinities of (RS)-NS1209 at the Glu....... The thermodynamics of binding of the antagonists (S)-NS1209, DNQX and (S)-ATPO to the GluR2 ligand-binding core have been determined by displacement isothermal titration calorimetry. The displacement of (S)-glutamate by all antagonists was shown to be driven by enthalpy....

  10. The Lepidoptera Odorant Binding Protein gene family: Gene gain and loss within the GOBP/PBP complex of moths and butterflies.

    Science.gov (United States)

    Vogt, Richard G; Große-Wilde, Ewald; Zhou, Jing-Jiang

    2015-07-01

    Butterflies and moths differ significantly in their daily activities: butterflies are diurnal while moths are largely nocturnal or crepuscular. This life history difference is presumably reflected in their sensory biology, and especially the balance between the use of chemical versus visual signals. Odorant Binding Proteins (OBP) are a class of insect proteins, at least some of which are thought to orchestrate the transfer of odor molecules within an olfactory sensillum (olfactory organ), between the air and odor receptor proteins (ORs) on the olfactory neurons. A Lepidoptera specific subclass of OBPs are the GOBPs and PBPs; these were the first OBPs studied and have well documented associations with olfactory sensilla. We have used the available genomes of two moths, Manduca sexta and Bombyx mori, and two butterflies, Danaus plexippus and Heliconius melpomene, to characterize the GOBP/PBP genes, attempting to identify gene orthologs and document specific gene gain and loss. First, we identified the full repertoire of OBPs in the M. sexta genome, and compared these with the full repertoire of OBPs from the other three lepidopteran genomes, the OBPs of Drosophila melanogaster and select OBPs from other Lepidoptera. We also evaluated the tissue specific expression of the M. sexta OBPs using an available RNAseq databases. In the four lepidopteran species, GOBP2 and all PBPs reside in single gene clusters; in two species GOBP1 is documented to be nearby, about 100 kb from the cluster; all GOBP/PBP genes share a common gene structure indicating a common origin. As such, the GOBP/PBP genes form a gene complex. Our findings suggest that (1) the lepidopteran GOBP/PBP complex is a monophyletic lineage with origins deep within Lepidoptera phylogeny, (2) within this lineage PBP gene evolution is much more dynamic than GOBP gene evolution, and (3) butterflies may have lost a PBP gene that plays an important role in moth pheromone detection, correlating with a shift from

  11. Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting.

    Science.gov (United States)

    Swainsbury, David J K; Martin, Elizabeth C; Vasilev, Cvetelin; Parkes-Loach, Pamela S; Loach, Paul A; Neil Hunter, C

    2017-11-01

    The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  12. New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

    Science.gov (United States)

    Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

    2014-03-03

    New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  13. Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of the Human Origin Recognition Complex

    National Research Council Canada - National Science Library

    Mendez, Juan

    2000-01-01

    ... of cellular life tipically lost in cancer. In order to unravel the molecular mechanisms of human DNA replication in normal and cancer cells, we have started a search for human DNA sequences that serve as replicators", this is, binding sites...

  14. A new signal-on method for the detection of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kai, E-mail: zhangkai@jsinm.org; Wang, Ke; Zhu, Xue; Xie, Minhao

    2015-08-05

    Proteins play important roles in biological and cellular processes. The levels of proteins can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of proteins is imperative to proteins express, study, and clinical diagnosis. Herein, we report a “signal-on” platform for the assay of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes. By using biotin as the affinity ligand, this simple protocol could sensitively detect streptavidin with a detection limit down to 10 pM. With the use of an antibody as the affinity ligand, a method for homogeneous fluorescence detection of Prostate Specific Antigen (PSA) was also proposed with a detection limit of 10 pM. The one-step and wash-free assay showed good selectivity. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis. - Highlights: • AgNCs have great potential for application in biomedicine. • Binding of two affinity ligands can result in binding-induced DNA assemblies. • PET can be happened between DNA/AgNCs and G-quadruplex/hemin complexes. • A platform for the detection of proteins was proposed by using PET and binding-induced strategy.

  15. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    Energy Technology Data Exchange (ETDEWEB)

    Ida, Tomoyo [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Suzuki, Hideyuki [Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Fukuyama, Keiichi [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Hiratake, Jun [Kyoto University, Uji, Kyoto 611-0011 (Japan); Wada, Kei, E-mail: keiwada@med.miyazaki-u.ac.jp [University of Miyazaki, Miyazaki 889-1692 (Japan); Osaka University, Toyonaka, Osaka 560-0043 (Japan)

    2014-02-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.

  16. Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid cleavage, cell imaging and cytotoxic studies.

    Science.gov (United States)

    Tabassum, Sartaj; Zaki, Mehvash; Ahmad, Musheer; Afzal, Mohd; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

    2014-08-18

    New Cu(II) complex 1 of indole-3-propionic acid and 1,10-phenanthroline was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. In vitro DNA binding studies of 1 was performed by employing UV-vis and fluorescence spectroscopic techniques. The binding affinity towards human serum albumin (HSA) was also investigated to understand the carrier role in body system, as the time dependent HPLC experiment of 1 revealed that bonded drug with protein releases slowly in presence of DNA. Complex 1 exhibited good anti-tumor activity (GI50 values <10 μg/ml), and to elucidate the mechanism of tumor inhibition, topoisomerase I enzymatic activity was carried out and further validated by cell imaging studies which clearly showed its nuclear localization. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. FSHR and LHR Expression and Signaling as Well as Maturation and Apoptosis of Cumulus-Oocyte Complexes Following Treatment with FSH Receptor Binding Inhibitor in Sheep

    Directory of Open Access Journals (Sweden)

    Suocheng Wei

    2017-09-01

    Full Text Available Background/Aims: Currently, it remains unknown whether FSH receptor binding inhibitor (FRBI influences follicular development and reproduction functions in humans and animals. The present study aimed to investigate FRBI effects on in vitro maturation (IVM and apoptosis of cumulus-oocyte complexes (COCs of sheep, to determine the effect of FRBI on mRNA and protein levels of FSHR and LHR in COCs, and to elucidate the signal pathway of FRBI effects. Methods: COCs were in vitro cultured for 24h in the IVM media supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40µg/mL and FSH (10IU/mL. The harvested COCs were observed under an inverted microscope and maturation rates of COCs were determined. Real time RT-PCR and Western blotting were utilized to detect mRNA and protein levels of FSHR and LHR. The concentrations of FSH, LH and caspase-3 were determined using especial ELISA kits for sheep, respectively. Results: Maturation rates of COCs decreased gradually as FRBI concentrations increased from 0 to 40µg/mL, reaching a bottom value of 23.76% of the FRBI-4 group. The maximal apoptosis rate was detected in the FRBI-4 group. IP3 contents of FRBI-3 and FRBI-4 groups were reduced as compared to control group (CG and FSH groups (P<0.05. Levels of FSHR protein of FRBI-3 and FRBI-4 groups as well as LHR protein of FRBI-4 group were significantly less than that of CG and FSH group. FSH contents of four FRBI treatment groups were gradually decreased along with the supplementation doses of FRBI. Caspase-3 contents of FRBI groups were reduced with a maximum reduction of the FRBI-2 group. Conclusion: Our results revealed supplement of FRBI into IVM media could dose-dependently decrease the maturation rate and increase apoptosis rate of sheep COCs. A lower dose of FRBI treatment slightly promoted IP3 production, but a higher dose of FRBI reduced IP3 production. FRBI suppressed the mRNA and protein expression levels of FSHR and LHR in sheep COCs

  18. The intermediate filament protein vimentin binds specifically to a recombinant integrin α2/β1 cytoplasmic tail complex and co-localizes with native α2/β1 in endothelial cell focal adhesions

    International Nuclear Information System (INIS)

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly

    2005-01-01

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen

  19. Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: Indication of a conformational change in the central helix

    International Nuclear Information System (INIS)

    Ikura, Mitsuhiko; Kay, L.E.; Bax, A.; Krinks, M.

    1991-01-01

    Heteronuclear 3D and 4D NMR experiments have been used to obtain 1 H, 13 C, and 15 N backbone chemical shift assignments in Ca 2+ -loaded clamodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-bionding domain (residues 577-602) of rabbit skeletal muscle muosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca 2+ -binding site 1 (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca 2+ -binding site 4 (F141-M145). Analysis of backbone NOE connectivities indicates a change from α-helical to an extended conformation for residues 75-77 upon complexation with M13. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site 3

  20. Regular square planer bis-(4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione)/copper(II) complex: Trans/cis-DFT isomerization, crystal structure, thermal, solvatochromism, hirshfeld surface and DNA-binding analysis

    Science.gov (United States)

    Hema, M. K.; Karthik, C. S.; Warad, Ismail; Lokanath, N. K.; Zarrouk, Abdelkader; Kumara, Karthik; Pampa, K. J.; Mallu, P.

    2018-04-01

    Trans-[Cu(O∩O)2] complex, O∩O = 4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione was reported with high potential toward CT-DNA binder. The solved XRD-structure of complex indicated a perfect regular square-planer geometry around the Cu(II) center. The trans/cis-DFT-isomerization calculation supported the XRD seen in reflecting the trans-isomer as the kinetic-favor isomer. The desired complex structure was also characterized by conductivity measurement, CHN-elemental analyses, MS, EDX, SEM, UV-Vis., FT-IR, HAS and TG/DTG. The Solvatochromism behavior of the complex was evaluated using four different polar solvents. MPE and Hirshfeld surface analysis (HSA) come to an agreement that fluoride and thiophene protons atoms are with suitable electro-potential environment to form non-classical H-bonds of type CThsbnd H⋯F. The DNA-binding properties were investigated by viscosity tests and spectrometric titrations, the results revealed the complex as strong calf-thymus DNA binder. High intrinsic-binding constants value ∼1.8 × 105 was collected.

  1. CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

    Energy Technology Data Exchange (ETDEWEB)

    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L. [Case Western; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

  2. Inefficient binding of IgM immune complexes to erythrocyte C3b-C4b receptors (CR1) and weak incorporation of C3b-iC3b into the complexes

    DEFF Research Database (Denmark)

    Kávai, M; Rasmussen, J M; Baatrup, G

    1988-01-01

    , but the binding was low (2-3%) when compared to the binding of the corresponding IgG-IC (50-60%). Solid phase IC were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37 degrees C....... The binding of C3b-iC3b was determined by use of biotinylated F(ab')2 antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b-iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching...

  3. Properties of the manganese(II) binding site in ternary complexes of Mnter dot ADP and Mnter dot ATP with chloroplast coupling factor 1: Magnetic field dependence of solvent sup 1 H and sup 2 H NMR relaxation rates

    Energy Technology Data Exchange (ETDEWEB)

    Haddy, A.E.; Frasch, W.D.; Sharp, R.R. (Univ. of Michigan, Ann Arbor (USA))

    1989-05-02

    The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF{sub 1}) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF{sub 1} under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl{sub 2} and removal of labile ligands by extensive gel filtration chromatography. In the field-dependent NMR experiments, the Mn(II) binding site of CF{sub 1} was studied for three mole ratios of added Mn(II) to CF{sub 1}, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of Cf{sub 1}. In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF{sub 1} were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded to bound Mn(II) increased from one in the binary Mn(II)-CF{sub 1} complex to three and two in the ternary Mn(II)-ADP-CF{sub 1} and Mn(II)-ATP-CF{sub 1} complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF{sub 1} complex is replaced by the {gamma}-phosphate of ATP in the Mn(II)-ATP-CF{sub 1} complex. A binding model is presented to account for these observations.

  4. Cellular expression or binding of desLys58-beta2 microglobulin is not dependent on the presence of the tri-molecular MHC class I complex

    DEFF Research Database (Denmark)

    Wang, M; Corlin, D B; Heegaard, N H H

    2008-01-01

    -exposure to dbeta2m. Binding of 332-01 antibody could not be displaced by addition of high concentrations of native beta2m. In conclusion, our data indicate that dbeta2m - in contrast to native beta2m - binds to a hitherto unknown cell surface receptor independent of classical MHC class I molecules. As beta2m has...... previously been shown to display biological activities such as the induction of both growth promotion and apoptosis, C1 complement activity, shown to mediate cleavage of beta2m, could be involved in these processes....

  5. A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Schoch, Angela; Larraillet, Vincent

    2017-01-01

    The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism...... half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct...

  6. A critical look at the calculation of the binding characteristics and concentration of iron complexing ligands in seawater with suggested improvements

    NARCIS (Netherlands)

    Gerringa, L.J.A.; Rijkenberg, M.J.A.; Thuróczy, C.-E.; Maas, L.R.M.

    2014-01-01

    Environmental context
    The low concentration of iron in the oceans limits growth of phytoplankton. Dissolved organic molecules, called ligands, naturally present in seawater, bind iron thereby increasing its solubility and, consequently, its availability for biological uptake by phytoplankton. The

  7. (Carboxydiamine)Pt(II) complexes of a combretastatin A-4 analogous chalcone: the influence of the diamine ligand on DNA binding and anticancer effects

    Czech Academy of Sciences Publication Activity Database

    Zoldakova, M.; Biersack, B.; Kostrhunová, Hana; Ahmad, A.; Padhye, S.; Sarkar, F.H.; Schobert, R.; Brabec, Viktor

    2011-01-01

    Roč. 2, č. 6 (2011), s. 493-499 ISSN 2040-2503 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : platinum * DNA binding * anticancer effects Subject RIV: BO - Biophysics Impact factor: 2.800, year: 2011

  8. The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI{sub 6} octahedral complex to a PtI{sub 3} moiety bound to His15

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M.; Starkey, Laurina-Victoria; Lamplough, Lucinda; Kaenket, Surasek; Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    The platinum hexahalides have an octahedral arrangement of six halogen atoms bound to a Pt centre, thus having an octahedral shape that could prove to be useful in interpreting poor electron-density maps. In a detailed characterization, PtI{sub 6} chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15 of HEWL was also observed, which was not observed for PtBr{sub 6} or PtCl{sub 6}. This study examines the binding and chemical stability of the platinum hexahalides K{sub 2}PtCl{sub 6}, K{sub 2}PtBr{sub 6} and K{sub 2}PtI{sub 6} when soaked into pre-grown hen egg-white lysozyme (HEWL) crystals as the protein host. Direct comparison of the iodo complex with the chloro and bromo complexes shows that the iodo complex is partly chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15, a chemical behaviour that is not exhibited by the chloro or bromo complexes. Each complex does, however, bind to HEWL in its octahedral form either at one site (PtI{sub 6}) or at two sites (PtBr{sub 6} and PtCl{sub 6}). As heavy-atom derivatives of a protein, the octahedral shape of the hexahalides could be helpful in cases of difficult-to-interpret electron-density maps as they would be recognisable ‘objects’.

  9. Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

    Science.gov (United States)

    Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

    2008-09-01

    We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

  10. Interactions of nickel(II) with histones. Stability and solution structure of complexes with CH3CO-Cys-Ala-Ile-His-NH2, a putative metal binding sequence of histone H3.

    Science.gov (United States)

    Bal, W; Lukszo, J; Jezowska-Bojczuk, M; Kasprzak, K S

    1995-01-01

    Nickel(II) compounds are established human carcinogens, but the molecular mechanisms underlying their activity are only partially known. One mechanism may include mediation by nickel of promutagenic oxidative DNA damage that depends on Ni(II) binding to chromatin. To characterize such binding at the histone moiety of chromatin, we synthesized the peptide CH3CO-Cys-Ala-Ile-His-NH2 (L), a model of the evolutionarily conserved motif in histone H3 with expected affinity for transition metals, and evaluated its reactivity toward Ni(II). Combined spectroscopic (UV/vis, CD, NMR) and potentiometric measurements showed that, at physiological pH, mixtures of Ni(II) and L yielded unusual macrochelate complexes, NiL and NiL2, in which the metal cation was bound through Cys and His side chains in a square-planar arrangement. Above pH 9, a NiH-3L complex was formed, structurally analogous to typical square-planar nickel complexes. These complexes are expected to catalyze oxidation reactions, and therefore, coordination of Ni(II) by the L motif in core histone H3 may be a key event in oxidative DNA base damage observed in the process of Ni(II)-induced carcinogenesis.

  11. A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

    Science.gov (United States)

    Choi, Eunji; Oh, Jungju; Lee, Dahee; Lee, Jaewon; Tan, Xiaonan; Kim, Minkyung; Kim, Gyeungyun; Piao, Chunxian; Lee, Minhyung

    2018-04-13

    The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Electrostatic interactions play an essential role in the binding of oleic acid with α-lactalbumin in the HAMLET-like complex: a study using charge-specific chemical modifications.

    Science.gov (United States)

    Xie, Yongjing; Min, Soyoung; Harte, Níal P; Kirk, Hannah; O'Brien, John E; Voorheis, H Paul; Svanborg, Catharina; Hun Mok, K

    2013-01-01

    Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Copyright © 2012 Wiley Periodicals, Inc.

  13. Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

    DEFF Research Database (Denmark)

    Laursen, Mette; Yatime, Laure; Nissen, Poul

    2013-01-01

    of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2–MgFx–ouabain structure [Ogawa et al...

  14. Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

    Science.gov (United States)

    Terai, Yuma; Sato, Ryuma; Yumiba, Takahiro; Harada, Ryuhei; Shimizu, Kohei; Toga, Tatsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Iwai, Shigenori; Shigeta, Yasuteru; Yamamoto, Junpei

    2018-05-14

    (6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

  15. Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein.

    Science.gov (United States)

    Shaikhali, Jehad; Davoine, Céline; Brännström, Kristoffer; Rouhier, Nicolas; Bygdell, Joakim; Björklund, Stefan; Wingsle, Gunnar

    2015-06-15

    The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combination with specific TFs as GeBPL. © The Authors Journal compilation © 2015 Biochemical Society.

  16. Synthesis, spectroscopic and DNA binding ability of CoII, NiII, CuII and ZnII complexes of Schiff base ligand (E)-1-(((1H-benzo[d]imidazol-2-yl)methylimino)methyl)naphthalen-2-ol. X-ray crystal structure determination of cobalt (II) complex.

    Science.gov (United States)

    Yarkandi, Naeema H; El-Ghamry, Hoda A; Gaber, Mohamed

    2017-06-01

    A novel Schiff base ligand, (E)-1-(((1H-benzo[d]imidazol-2-yl)methylimino)methyl)naphthalen-2-ol (HL), has been designed and synthesized in addition to its metal chelates [Co(L) 2 ]·l2H 2 O, [Ni(L)Cl·(H 2 O) 2 ].5H 2 O, [Cu(L)Cl] and [Zn(L)(CH 3 COO)]. The structures of the isolated compounds have been confirmed and identified by means of different spectral and physicochemical techniques including CHN analysis, 1 H & 13 C NMR, mass spectral analysis, molar conductivity measurement, UV-Vis, infrared, magnetic moment in addition to TGA technique. The infrared spectral results ascertained that the ligand acts as monobasic tridentate binding to the metal centers via deprotonated hydroxyl oxygen, azomethine and imidazole nitrogen atoms. The UV-Vis, magnetic susceptibility and molar conductivity data implied octahedral geometry for Co(II) & Ni(II) complexes, tetrahedral for Zn(II) complex and square planar for Cu(II) complex. X-ray structural analysis of Co(II) complex 1 has been reported and discussed. Moreover, the type of interaction between the ligand & its complexes towards salmon sperm DNA (SS-DNA) has been examined by the measurement of absorption spectra and viscosity which confirmed that the ligand and its complexes interact with DNA via intercalation interaction as concluded from the values of binding constants (K b ). Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Reynolds K Brobey

    Full Text Available The reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1 signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1 covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2 in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx proteins simultaneously engage in a tripartite complex formation; 3 Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

  18. Molecular dynamics-assisted pharmacophore modeling of caspase-3-isatin sulfonamide complex: Recognizing essential intermolecular contacts and features of sulfonamide inhibitor class for caspase-3 binding.

    Science.gov (United States)

    Kumar, Sivakumar Prasanth; Patel, Chirag N; Jha, Prakash C; Pandya, Himanshu A

    2017-12-01

    The identification of isatin sulfonamide as a potent small molecule inhibitor of caspase-3 had fuelled the synthesis and characterization of the numerous sulfonamide class of inhibitors to optimize for potency. Recent works that relied on the ligand-based approaches have successfully shown the regions of optimizations for sulfonamide scaffold. We present here molecular dynamics-based pharmacophore modeling of caspase-3-isatin sulfonamide crystal structure, to elucidate the essential non-covalent contacts and its associated pharmacophore features necessary to ensure caspase-3 optimal binding. We performed 20ns long dynamics of this crystal structure to extract global conformation states and converted into structure-based pharmacophore hypotheses which were rigorously validated using an exclusive focussed library of experimental actives and inactives of sulfonamide class by Receiver Operating Characteristic (ROC) statistic. Eighteen structure-based pharmacophore hypotheses with better sensitivity and specificity measures (>0.6) were chosen which collectively showed the role of pocket residues viz. Cys163 (S 1 sub-site; required for covalent and H bonding with Michael acceptor of inhibitors), His121 (S 1 ; π stack with bicyclic isatin moiety), Gly122 (S 1 ; H bond with carbonyl oxygen) and Tyr204 (S 2 ; π stack with phenyl group of the isatin sulfonamide molecule) as stringent binding entities for enabling caspase-3 optimal binding. The introduction of spatial pharmacophore site points obtained from dynamics-based pharmacophore models in a virtual screening strategy will be helpful to screen and optimize molecules belonging to sulfonamide class of caspase-3 inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Quantification of biopharmaceuticals and biomarkers in complex biological matrices: a comparison of liquid chromatography coupled to tandem mass spectrometry and ligand binding assays

    NARCIS (Netherlands)

    Bults, Peter; van de Merbel, Nico C; Bischoff, Rainer

    2015-01-01

    The quantification of proteins (biopharmaceuticals or biomarkers) in complex biological samples such as blood plasma requires exquisite sensitivity and selectivity, as all biological matrices contain myriads of proteins that are all made of the same 20 proteinogenic amino acids, notwithstanding

  20. Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2

    DEFF Research Database (Denmark)

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle

    2010-01-01

    We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely relate...... and prostate cancer.......We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related...... PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural...

  1. Size-dependent binding energies and fine-structure splitting of excitonic complexes in single InAs/GaAs quantum dots

    International Nuclear Information System (INIS)

    Rodt, S.; Seguin, R.; Schliwa, A.; Guffarth, F.; Poetschke, K.; Pohl, U.W.; Bimberg, D.

    2007-01-01

    A systematic study of excitonic complexes confined in single InAs/GaAs quantum dots is presented. Emphasis is placed on the recombination energies of the excitonic complexes and on the fine-structure splitting of the bright exciton ground state. The values depend in a characteristic way on the size of the respective quantum dot which controls the number of bound hole states and the piezoelectric potential

  2. High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP–oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

    Directory of Open Access Journals (Sweden)

    E. I. Howard

    2016-03-01

    Full Text Available Crystal diffraction data of heart fatty acid binding protein (H-FABP in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively. These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H...H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.

  3. Synthesis, spectroscopic, thermal and molecular modeling studies of Zn2+, Cd2+ and UO22+ complexes of Schiff bases containing triazole moiety. Antimicrobial, anticancer, antioxidant and DNA binding studies.

    Science.gov (United States)

    Gaber, Mohamed; El-Ghamry, Hoda A; Fathalla, Shaimaa K; Mansour, Mohammed A

    2018-02-01

    A novel series of Zn 2+ , Cd 2+ and UO 2 2+ complexes of ligands namely 1-[(5-mercapto-1H-1,2,4-triazole-3-ylimino) methyl]naphthalene-2-ol (HL 1 ) and [(1H-1,2,4-triazole-3-ylimino) methyl] naphthalene-2-ol (HL 2 ) have been prepared and characterized by different analytical and spectral techniques. The stoichiometry, stereochemistry, conductivity measurements and mode of bonding of the complexes have been elucidated. Accurate comparison of the IR spectra of the ligands with their metal chelates proved the involvement of nitrogen atoms of the azomethine group and/or triazole ring in chelation in addition to the deprotonated hydroxyl oxygen. The UV-Vis and molar conductance data supported the octahedral geometry for the metal complexes. TGA technique has been used to study the thermal decomposition way of the metal complexes and the thermo kinetic parameters were estimated. Valuable information is obtained from calculations of molecular parameters using the molecular modeling techniques. The interaction between the metal complexes and CT-DNA has been studied from which the binding constants (k b ) were calculated. The Schiff bases and their metal chelates have shown potent antimicrobial, antioxidant and antitumor activities. The antitumor activities of the compounds have been tested in vitro against HEPG2 cell line and in silico by the molecular docking analysis with the VEGFR-2 receptor responsible for angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

    Science.gov (United States)

    Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2008-01-01

    The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

  5. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2008-03-04

    The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

  6. A Ru(II) complex with 2-(4-(methylsulfonyl)phenyl)-1H-imidazo[4,5- f][1,10]phenanthroline: Synthesis, characterization, and acid-base and DNA-binding properties

    Science.gov (United States)

    Gao, Jie; Wang, Zhi-Ping; Yuan, Cui-Li; Jia, Hai-Shun; Wang, Ke-Zhi

    2011-09-01

    A new Ru(II) complex of [Ru(bpy) 2(Hmspip)]Cl 2 {in which bpy = 2,2'-bipyridine, Hmspip = 2-(4-(methylsulfonyl)phenyl)-1 H-imidazo[4,5- f][1,10]phenanthroline} have been synthesized and characterized. The ground- and excited-state acid-base properties of [Ru(bpy) 2(Hmspip)]Cl 2 and its parent complex of [Ru(bpy) 2(Hpip)]Cl 2 {Hpip = 2-phenyl-1H-imidazo[4,5- f][1,10]phenanthroline} have been studied by UV-visible (UV-vis) and emission spectrophotometric pH titrations. [Ru(bpy) 2(Hmspip)]Cl 2 acts as a calf thymus DNA intercalators with a binding constant of 4.0 × 10 5 M -1 in buffered 50 mM NaCl, as evidenced by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN) 6] 4-, DNA competitive binding with ethidium bromide, reverse salt titrations and viscosity measurements.

  7. Structure-guided approach identifies a novel class of HIV-1 ribonuclease H inhibitors: binding mode insights through magnesium complexation and site-directed mutagenesis studies

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Corona, Angela; Steinmann, Casper

    2018-01-01

    is a long and expensive process that can be speeded up by in silico methods. In the present study, a structure-guided screening is coupled with a similarity-based search on the Specs database to identify a new class of HIV-1 RNase H inhibitors. Out of the 45 compounds selected for experimental testing, 15...... inhibited the RNase H function below 100 μM with three hits exhibiting IC50 values active compound, AA, inhibits HIV-1 RNase H with an IC50 of 5.1 μM and exhibits a Mg-independent mode of inhibition. Site-directed mutagenesis studies provide valuable insight into the binding mode of newly...

  8. Low Levels of the 150-kD Insulin -Like Growth Factor Binding Protein 3 Ternary complex in Patients with Anorexia nervosa

    DEFF Research Database (Denmark)

    Støving, René K; Hangaard, Jørgen; Hagen, Claus

    2003-01-01

    women with AN at the time of diagnosis and after partial weight recovery and in 6 healthy age-matched women serving as controls. RESULTS: Patients with AN had low levels of ALS and IGFBP-3 contained in the 150-kD ternary complex and in the non-150-kD fraction. Following partial weight recovery, the 150......-kD IGFBP-3 ternary complex was fully normalized, despite only partial normalization of serum GH and IGF-I levels. Patients with AN did not present with IGFBP-3 proteolysis different from controls. CONCLUSION: The present data indicate a pivotal role of the nutritional status in the regulation...

  9. Metal based biologically active compounds: Design, synthesis, DNA binding and antidiabetic activity of 6-methyl-3-formyl chromone derived hydrazones and their metal (II) complexes.

    Science.gov (United States)

    Philip, Jessica Elizabeth; Shahid, Muhammad; Prathapachandra Kurup, M R; Velayudhan, Mohanan Puzhavoorparambil

    2017-10-01

    Two chromone hydrazone ligands HL 1 and HL 2 were synthesized and characterized by elemental analyses, IR, 1 H NMR & 13 C NMR, electronic absorption and mass spectra. The reactions of the chromone hydrazones with transition metals such as Ni, Cu, and Zn (II) salts of acetate afforded mononuclear metal complexes. Characterization and structure elucidation of the prepared chromone hydrazone metal (II) complexes were done by elemental, IR, electronic, EPR spectra and thermo gravimetric analyses as well as conductivity and magnetic susceptibility measurements. The spectroscopic data showed that the ligand acts as a mono basic bidentate with coordination sites are azomethine nitrogen and hydrazonic oxygen, and they exhibited distorted geometry. The biological studies involved antidiabetic activity i.e. enzyme inhibition of α-amylase and α-glucosidase, Calf Thymus - DNA (CT-DNA) interaction and molecular docking. Potential capacity of synthesized compounds to inhibit the α-amylase and α-glucosidase activity was assayed whereas DNA interaction studies were carried out with the help UV-Vis absorption titration and viscosity method. The docking studies of chromone hydrazones show that they are minor groove binders. Complexes were found to be good DNA - intercalates. Chromone hydrazones and its transition metal complexes have shown comparable antidiabetic activity with a standard drug acarbose. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Photoprotection in Plants Involves a Change in Lutein 1 Binding Domain in the Major Light-harvesting Complex of Photosystem II

    NARCIS (Netherlands)

    Ilioaia, C.; Johnson, M.P.; Liao, P.N.; Pascal, A.A.; van Grondelle, R.; Walla, P.J.; Ruban, A.V.; Robert, B.

    2011-01-01

    Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem

  11. Synthesis, spectroscopic characterization and structural investigations of a new charge transfer complex of 2,6-diaminopyridine with 3,5-dinitrobenzoic acid: DNA binding and antimicrobial studies

    Science.gov (United States)

    Khan, Ishaat M.; Ahmad, Afaq; Kumar, Sarvendra

    2013-03-01

    A new charge transfer (CT) complex [(DAPH)+(DNB)-] consisting of 2,6-diaminopyridine (DAP) as donor and 3,5-dinitrobenzoic acid (DNB-H) as acceptor, was synthesized and characterized by FTIR, 1H and 13C NMR, ESI mass spectroscopic and X-ray crystallographic techniques. The hydrogen bonding (N+-H⋯O-) plays an important role to consolidate the cation and anion together. CT complex shows a considerable interaction with Calf thymus DNA. The CT complex was also tested for its antibacterial activity against two Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and two Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa strains by using Tetracycline as standard, and antifungal property against Aspergillus niger, Candida albicans, and Penicillium sp. by using Nystatin as standard. The results were compared with standard drugs and significant conclusions were obtained. A polymeric net work through H-bonding interactions between neighboring moieties was observed. This has been attributed to the formation of 1:1 type CT complex.

  12. Synthesis, characterization, and DNA binding and cleavage properties of copper(II)-tryptophanphenyl-alanine-1,10-phenanthroline/2,2'-bipyridine complexes.

    Science.gov (United States)

    Reddy, Pulimamidi R; Raju, Nomula; Satyanarayana, Battu

    2011-01-01

    The mononuclear dipeptide-based Cu(II) complexes [Cu(II) (trp-phe)(phen)(H₂O)] ⋅ ClO₄ (1) and [Cu(II) (trp-phe)(bpy)(H₂O)] ⋅ ClO₄ (2) (trp-phe=tryptophanphenylalanine, phen=1,10-phenanthroline, bpy=2,2'-bipyridine) were isolated, and their interaction with DNA was studied. They exhibit intercalative mode of interaction with DNA. The intercalative interaction was quantified by Stern-Volmer quenching constant (K(sq) =0.14 for 1 and 0.08 for 2). The Cu(II) complexes convert supercoiled plasmid DNA into its nicked circular form hydrolytically at physiological conditions at a concentration as low as 5 μM (for 1) and 10 μM (for 2). The DNA hydrolysis rates at a complex concentration of 50 μM were determined as 1.74 h(-1) (R=0.985) for 1 and 0.65 h(-1) (R=0.965) for 2. The rate enhancement in the range of 2.40-4.10×10⁷-fold compared to non-catalyzed double-stranded DNA is significant. This was attributed to the presence of a H(2) O molecule in the axial position of the Cu complexes. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.

  13. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

    DEFF Research Database (Denmark)

    Vorup-Jensen, T; Petersen, Steen Vang; Hansen, A G

    2000-01-01

    proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution. Udgivelsesdato: 2000...

  14. Synthesis, characterization, DNA-binding study and anticancer properties of ternary metal(II) complexes of edda and an intercalating ligand.

    Science.gov (United States)

    Ng, Chew Hee; Kong, King Chow; Von, Sze Tin; Balraj, Pauline; Jensen, Paul; Thirthagiri, Eswary; Hamada, Hirokazu; Chikira, Makoto

    2008-01-28

    A series of ternary metal(ii) complexes {M(phen)(edda); 1a (Cu), 1b (Co), 1c (Zn), 1d (Ni); H(2)edda = N,N(')-ethylenediaminediacetic acid} of N,N'-ethylene-bridged diglycine and 1,10-phenanthroline were synthesized and characterized by elemental analysis, FTIR, UV-visible spectroscopy and magnetic susceptibility measurement. The interaction of these complexes with DNA was investigated using CD and EPR spectroscopy. MTT assay results of 1a-1c , screened on MCF-7 cancer cell lines, show that synergy between the metal and ligands results in significant enhancement of their antiproliferative properties. Preliminary results from apoptosis and cell cycle analyses with flow cytometry are reported. seems to be able to induce cell cycle arrest at G(0)/G(1). The crystal structure of 1a is also included.

  15. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2001-01-01

    Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified...

  16. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

    Science.gov (United States)

    Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

    2012-02-14

    Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Capturing the photo-signaling state of a photoreceptor in a steady-state fashion by binding a transition metal complex.

    Science.gov (United States)

    Yu, Pengyun; Song, Lei; Qin, Jun; Wang, Jianping

    2017-11-01

    Binding a small molecule to proteins causes conformational changes, but often to a limited extent. Here, we demonstrate that the interaction of a CO-releasing molecule (CORM3) with a photoreceptor photoactive yellow protein (PYP) drives large structural changes in the latter. The interaction of CORM3 and a mutant of PYP, Met100Ala, not only trigger the isomerization of its chromophore, p-coumaric acid, from its anionic trans configuration to a protonated cis configuration, but also increases the content of β-sheet at the cost of α-helix and random coil in the secondary structure of the protein. The CORM3 derived Met100Ala is found to highly resemble the signaling state, which is one of the key photo-intermediates of this photoactive protein, in both protein local conformation and chromophore configuration. The organometallic reagents hold promise as protein engineering tools. This work highlights a novel approach to structurally accessing short lived intermediates of proteins in a steady-state fashion. © 2017 The Protein Society.

  18. Host-Guest Complexes of Cyclodextrins and Nanodiamonds as a Strong Non-Covalent Binding Motif for Self-Assembled Nanomaterials.

    Science.gov (United States)

    Schibilla, Frauke; Voskuhl, Jens; Fokina, Natalie A; Dahl, Jeremy E P; Schreiner, Peter R; Ravoo, Bart Jan

    2017-11-13

    We report the inclusion of carboxy- and amine-substituted molecular nanodiamonds (NDs) adamantane, diamantane, and triamantane by β-cyclodextrin and γ-cyclodextrin (β-CD and γ-CD), which have particularly well-suited hydrophobicity and symmetry for an optimal fit of the host and guest molecules. We studied the host-guest interactions in detail and generally observed 1:1 association of the NDs with the larger γ-CD cavity, but observed 1:2 association for the largest ND in the series (triamantane) with β-CD. We found higher binding affinities for carboxy-substituted NDs than for amine-substituted NDs. Additionally, cyclodextrin vesicles (CDVs) were decorated with d-mannose by using adamantane, diamantane, and triamantane as non-covalent anchors, and the resulting vesicles were compared with the lectin concanavalin A in agglutination experiments. Agglutination was directly correlated to the host-guest association: adamantane showed lower agglutination than di- or triamantane with β-CDV and almost no agglutination with γ-CDV, whereas high agglutination was observed for di- and triamantane with γ-CDV. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. In vivo binding and autoradiographic imaging of (+)-3-[125I]Iodo-MK-801 to the NMDA receptor-channel complex in rat brain

    International Nuclear Information System (INIS)

    Gibson, R.E.; Burns, H.D.; Thorpe, H.H.; Waisi Eng; Ransom, R.; Solomon, H.

    1992-01-01

    Radioiodinated (+)-3-Iodo-MK-801 is a high affinity radioligand for the N-methyl-D-aspartate (NMDA) receptor-channel complex. We have demonstrated in vivo localization in the CNS of rat which is stereoselective and blocked by coinjection of unlabeled MK-801. Autoradiography indicates localization in vivo which is in concordance with in vitro autoradiographic studies. These results indicate that radioiodinated (+)-3-Iodo-MK-801 is a useful probe for in vitro and in vivo autoradiographic studies and suggest that radioligands for the NMDA receptor may be developed which will provide in vivo images of receptor distribution in man. (author)

  20. Synthesis, spectroscopic and DNA binding ability of Co{sup II}, Ni{sup II}, Cu{sup II} and Zn{sup II} complexes of Schiff base ligand (E)-1-(((1H-benzo[d]imidazol-2-yl)methylimino)methyl)naphthalen-2-ol. X-ray crystal structure determination of cobalt (II) complex

    Energy Technology Data Exchange (ETDEWEB)

    Yarkandi, Naeema H. [Chemistry Department, Faculty of Applied Science, Umm Al–Qura University, Makkah (Saudi Arabia); El-Ghamry, Hoda A., E-mail: helghamrymo@yahoo.com [Chemistry Department, Faculty of Applied Science, Umm Al–Qura University, Makkah (Saudi Arabia); Chemistry Department, Faculty of Science, Tanta University, Tanta (Egypt); Gaber, Mohamed [Chemistry Department, Faculty of Science, Tanta University, Tanta (Egypt)

    2017-06-01

    A novel Schiff base ligand, (E)-1-(((1H-benzo[d]imidazol-2-yl)methylimino)methyl)naphthalen-2-ol (HL), has been designed and synthesized in addition to its metal chelates [Co(L){sub 2}]·l2H{sub 2}O, [Ni(L)Cl·(H{sub 2}O){sub 2}].5H{sub 2}O, [Cu(L)Cl] and [Zn(L)(CH{sub 3}COO)]. The structures of the isolated compounds have been confirmed and identified by means of different spectral and physicochemical techniques including CHN analysis, {sup 1}H &{sup 13}C NMR, mass spectral analysis, molar conductivity measurement, UV–Vis, infrared, magnetic moment in addition to TGA technique. The infrared spectral results ascertained that the ligand acts as monobasic tridentate binding to the metal centers via deprotonated hydroxyl oxygen, azomethine and imidazole nitrogen atoms. The UV–Vis, magnetic susceptibility and molar conductivity data implied octahedral geometry for Co(II) & Ni(II) complexes, tetrahedral for Zn(II) complex and square planar for Cu(II) complex. X-ray structural analysis of Co(II) complex 1 has been reported and discussed. Moreover, the type of interaction between the ligand & its complexes towards salmon sperm DNA (SS-DNA) has been examined by the measurement of absorption spectra and viscosity which confirmed that the ligand and its complexes interact with DNA via intercalation interaction as concluded from the values of binding constants (K{sub b}). - Highlights: • Synthesis of Co{sup II}, Ni{sup II}, Cu{sup II} and Zn{sup II} complexes of the Schiff base ligand based on 2-(aminomethyl)benzimidazole moiety. • The constitutions and structures of the ligand and complexes were elucidated. • Molecular structure of Co{sup II} complex was confirmed by single crystal X-ray diffraction method. • The ligand and its complexes interact with SS-DNA via intercalation mods.

  1. TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element.

    Directory of Open Access Journals (Sweden)

    Thomas Spaller

    Full Text Available The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

  2. Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

    Science.gov (United States)

    Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

    2007-01-01

    Quantitative structure-activity relationship (QSAR) a