WorldWideScience

Sample records for binding site mutants

  1. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

  2. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A;

    1999-01-01

    Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV......) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary...... to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly...

  3. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    Science.gov (United States)

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  4. HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants.

    Science.gov (United States)

    Maček Hrvat, Nikolina; Žunec, Suzana; Taylor, Palmer; Radić, Zoran; Kovarik, Zrinka

    2016-11-25

    The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms.

  5. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

    OpenAIRE

    Wang, Hui; Gouaux, Eric

    2012-01-01

    The controversy about the number of substrate binding sites in the neurotransmitter sodium symporter model protein LeuT is ongoing. Ligand binding assays and crystal structures of the LeuT F253A mutant support the presence of a single high affinity substrate binding site.

  6. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

    Science.gov (United States)

    Veitch, N C; Tams, J W; Vind, J; Dalbøge, H; Welinder, K G

    1994-06-15

    Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

  7. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue.

    Science.gov (United States)

    Wang, Hui; Gouaux, Eric

    2012-09-01

    LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

  8. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Gouaux, Eric (Oregon HSU)

    2012-10-10

    LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism ofr LeuT that involves a single high-affinity substrate binding site.

  9. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    Science.gov (United States)

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  10. Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.

    Science.gov (United States)

    Ghatge, Mohini S; Karve, Sayali S; David, Tanya M S; Ahmed, Mostafa H; Musayev, Faik N; Cunningham, Kendra; Schirch, Verne; Safo, Martin K

    2016-05-01

    Pyridoxal 5'-phosphate (PLP) is a cofactor for many vitamin B6-requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5'-phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE). This disorder has no effective treatment, and is often fatal unless treated with PLP. In this study, we show that R95C hPNPO exhibits a 15-fold reduction in affinity for the FMN cofactor, a 71-fold decrease in affinity for the substrate PNP, a 4.9-fold decrease in specific activity, and a 343-fold reduction in catalytic activity, compared to the wild-type enzyme. We have reported similar findings for R229W hPNPO. This report also shows that wild-type, R95C and R229W hPNPO bind PLP tightly at a noncatalytic site and transfer it to activate an apo-B6 enzyme into the catalytically active holo-form. We also show for the first time that hPNPO forms specific interactions with several B6 enzymes with dissociation constants ranging from 0.3 to 12.3 μm. Our results suggest a possible in vivo role for the tight binding of PLP in hPNPO, whether wild-type or variant, by protecting the very reactive PLP, and transferring this PLP directly to activate apo-B6 enzymes.

  11. Copper(II) complexes of terminally free alloferon mutants containing two histidyl binding sites inside peptide chain structure and stability.

    Science.gov (United States)

    Kadej, Agnieszka; Kuczer, Mariola; Kowalik-Jankowska, Teresa

    2015-12-21

    Mononuclear and polynuclear copper(II) complexes of alloferon 1 with point mutations, H1A/H12A H2N-A(1)GVSGH(6)GQH(9)GVA(12)G-COOH, H1A/H9A H2N-A(1)GVSGH(6)GQA(9)GVH(12)G-COOH, and H1A/H6A H2N-A(1)GVSGA(6)GQH(9)GVH(12)G-COOH, have been studied by potentiometric, UV-visible, CD, and EPR spectroscopy, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand molar ratios ranging from 1 : 1 to 3 : 1 was obtained. Over a wide 6-8 pH range, including physiological pH 7.4, and a 1 : 1 metal-to-ligand molar ratio, the peptides studied formed a CuH-1L complex with the 4N{NH2,N(-),2NIm} coordination mode. The presence of the 4N binding site for the CuH-1L complexes prevented the deprotonation and coordination of the second amide nitrogen atom to copper(II) ions (pK-1/-2 7.83-8.07) compared to that of pentaGly (6.81). The amine nitrogen donor and two imidazole nitrogen atoms (H(6)H(9), H(6)H(12) and H(9)H(12)) can be considered to be independent metal-binding sites in the species formed. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2 : 1 and 3 : 1 molar ratios dominate in the solution, respectively. For the Cu(II)-H1A/H9A and Cu(II)-H1A/H12A systems, the Cu3H-9L complexes are likely formed by the coordination of amide nitrogen atoms towards C-termini with ring sizes (7,5,5).

  12. An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

    Science.gov (United States)

    Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

    2009-10-25

    The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

  13. Swedish mutant APP-based BACE1 binding site peptide reduces APP β-cleavage and cerebral Aβ levels in Alzheimer's mice.

    Science.gov (United States)

    Li, Song; Hou, Huayan; Mori, Takashi; Sawmiller, Darrell; Smith, Adam; Tian, Jun; Wang, Yanjiang; Giunta, Brian; Sanberg, Paul R; Zhang, Sheqing; Tan, Jun

    2015-06-19

    BACE1 initiates amyloid-β (Aβ) generation and the resultant cerebral amyloidosis, as a characteristic of Alzheimer's disease (AD). Thus, inhibition of BACE1 has been the focus of a large body of research. The most recent clinical trials highlight the difficulty involved in this type of anti-AD therapy as evidenced by side effects likely due to the ubiquitous nature of BACE1, which cleaves multiple substrates. The human Swedish mutant form of amyloid protein precursor (APPswe) has been shown to possess a higher affinity for BACE1 compared to wild-type APP (APPwt). We pursued a new approach wherein harnessing this greater affinity to modulate BACE1 APP processing activity. We found that one peptide derived from APPswe, containing the β-cleavage site, strongly inhibits BACE1 activity and thereby reduces Aβ production. This peptide, termed APPswe BACE1 binding site peptide (APPsweBBP), was further conjugated to the fusion domain of the HIV-1 Tat protein (TAT) at the C-terminus to facilitate its biomembrane-penetrating activity. APPwt and APPswe over-expressing CHO cells treated with this TAT-conjugated peptide resulted in a marked reduction of Aβ and a significant increase of soluble APPα. Intraperitoneal administration of this peptide to 5XFAD mice markedly reduced β-amyloid deposits as well as improved hippocampal-dependent learning and memory.

  14. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    Science.gov (United States)

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  15. In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond.

    Science.gov (United States)

    Odaka, M; Kiribuchi, K; Allison, W S; Yoshida, M

    1993-12-27

    When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

  16. Aberrant cGMP-binding activity in non-chemotactic Dictyostelium discoideum mutants

    NARCIS (Netherlands)

    Kuwayama, Hidekazu; Viel, Gerhard T.; Ishida, Shuji; Haastert, Peter J.M. van

    1995-01-01

    The kinetics of cGMP-binding to the major cGMP-binding activity in Dictyostelium, were investigated in 10 non-chemotactic mutants (KI mutants; KI-1 similar to 10). A wild-type cell contains about 3000 binding sites with a K-d of 1.5 nM. cGMP may dissociate from these binding sites with fast (F-type)

  17. In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth.

    Science.gov (United States)

    Ross, Leila S; Gamo, Francisco Javier; Lafuente-Monasterio, Maria José; Singh, Onkar M P; Rowland, Paul; Wiegand, Roger C; Wirth, Dyann F

    2014-06-27

    Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

  18. Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

    Science.gov (United States)

    Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

    2013-12-27

    For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

  19. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

    1989-01-01

    : DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6...

  20. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding.

    Science.gov (United States)

    Arden, Susan D; Tumbarello, David A; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-10-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

  1. An additional substrate binding site in a bacterial phenylalanine hydroxylase.

    Science.gov (United States)

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Corn, Isaac R; Wagner, Kyle T; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2013-09-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.

  2. Chloride binding site of neurotransmitter sodium symporters.

    Science.gov (United States)

    Kantcheva, Adriana K; Quick, Matthias; Shi, Lei; Winther, Anne-Marie Lund; Stolzenberg, Sebastian; Weinstein, Harel; Javitch, Jonathan A; Nissen, Poul

    2013-05-21

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

  3. Engineering and Directed Evolution of a Ca2+ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu75–Leu82 to Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Eliel R. Romero-García

    2009-01-01

    G102D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects β-sheet e3 with α-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.

  4. Characterization of a Deswapped Triple Mutant Bovine Odorant Binding Protein

    Directory of Open Access Journals (Sweden)

    Roberto Favilla

    2011-04-01

    Full Text Available The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

  5. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  6. Statistics for Transcription Factor Binding Sites

    OpenAIRE

    2008-01-01

    Transcription factors (TFs) play a key role in gene regulation. They interact with specific binding sites or motifs on the DNA sequence and regulate expression of genes downstream of these binding sites. In silico prediction of potential binding of a TF to a binding site is an important task in computational biology. From a statistical point of view, the DNA sequence is a long text consisting of four different letters ('A','C','G', and 'T'). The binding of a TF to the sequence corresponds to ...

  7. Modulation of RNase E activity by alternative RNA binding sites.

    Directory of Open Access Journals (Sweden)

    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  8. Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity.

    Science.gov (United States)

    Pepper, Lauren R; Hammer, Daniel A; Boder, Eric T

    2006-06-30

    Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.

  9. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

  10. Microbes bind complement inhibitor factor H via a common site.

    Directory of Open Access Journals (Sweden)

    T Meri

    Full Text Available To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH. FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20. We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii. We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."

  11. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  12. Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes†

    OpenAIRE

    Manieri, Wanda; Moore, Molly E.; Soellner, Matthew B.; Tsang, Pearl; Caperelli, Carol A.

    2007-01-01

    Human glycinamide ribonucleotide transformylase (GART) (EC2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, D144, and the semi-conserved K170 in substrate binding and catalysis. Only two conservative substitutions, N...

  13. SERCA mutant E309Q binds two Ca ions but adopts a catalytically incompetent conformation

    DEFF Research Database (Denmark)

    Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand;

    2013-01-01

    The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross-talk between the two Ca2+ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular...... signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu309 contributes to Ca2+ coordination at site II, and a consensus has been that E309Q only binds Ca2+ at site I. The crystal structure of E309Q in the presence of Ca2+ and an ATP analogue, however, reveals two...... occupied Ca2+ sites of a non-catalytic Ca2E1 state. Ca2+ is bound with micromolar affinity by both Ca2+ sites in E309Q, but without cooperativity. The Ca2+-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring...

  14. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Bernhard [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany); Lecker, Laura S. M.; Zoltner, Martin [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Jaenicke, Elmar [Johannes Gutenberg-Universität, Jakob Welder Weg 26, 55128 Mainz (Germany); Schnell, Robert [Karolinska Institutet, 17 177 Stockholm (Sweden); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Brenk, Ruth, E-mail: w.n.hunter@dundee.ac.uk [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany)

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  15. Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H(1) and H(6) or H(9) or H(12)) binding sites Structure Stability and Biological Activity.

    Science.gov (United States)

    Matusiak, Agnieszka; Kuczer, Mariola; Czarniewska, Elżbieta; Urbański, Arkadiusz; Rosiński, Grzegorz; Kowalik-Jankowska, Teresa

    2015-10-01

    Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

  16. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh (Michigan)

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  17. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  18. Tissue specificity of endothelin binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Bolger, G.T.; Liard, F.; Krogsrud, R.; Thibeault, D.; Jaramillo, J. (BioMega, Inc., Laval, Quebec (Canada))

    1990-09-01

    A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.

  19. Predicted metal binding sites for phytoremediation.

    Science.gov (United States)

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-09-05

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do not appear in the essential oil and that some of these species are able to grow in metal contaminated sites. A pattern search against the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases yielded true positives in each case showing the high specificity of the motifs designed for the ions of nickel, lead, molybdenum, manganese, cadmium, zinc, iron, cobalt and xenobiotic compounds. Motifs were also studied against PDB structures. Results of the study suggested the presence of binding sites on the surface of protein molecules involved. PDB structures of proteins were finally predicted for the binding sites functionality in their respective phytoremediation usage. This was further validated through CASTp server to study its physico-chemical properties. Bioinformatics implications would help in designing strategy for developing transgenic plants with increased metal binding capacity. These metal binding factors can be used to restrict metal update by plants. This helps in reducing the possibility of metal movement into the food chain.

  20. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  1. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  2. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    Science.gov (United States)

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-01-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.

  3. The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

    Science.gov (United States)

    Tseng, C C; Miyamoto, M; Ramalingam, K; Hemavathy, K C; Levine, M J; Ramasubbu, N

    1999-02-01

    Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type

  4. Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Calmodulin (CaM) is a multifunctional Ca2+-binding protein regulating the activity of many enzymes in response to fluctuation of the intracellular Ca2+ level. It has been shown that a CaM Q41C/K75C mutant (CaMSS) with a disulfide bond in the N-terminal domain exhibits greatly reduced affinity to Ca2+. In the present study, the experimental results revealed a unique metal binding pattern in CaMSS towards La3+ and Ca2+ separately: the mutant protein binds Ca2+ at site Ⅰ, Ⅲ and IV; however, it binds La3+ at site Ⅰ, Ⅱ and IV. A putative mechanism was proposed which is the conformation of site Ⅱ (or siteⅢ) of CaMSS could be altered and thus loses its metal ion affinity in response to metal binding in the opposite terminal domain possibly through the long range domain interaction. The present work may offer new perspectives for understanding the mechanisms of specific metal ion affinity in CaM and for CaM-based protein design.

  5. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...... have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion...

  6. Computational identification of uncharacterized cruzain binding sites.

    Directory of Open Access Journals (Sweden)

    Jacob D Durrant

    Full Text Available Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention.

  7. Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

    Science.gov (United States)

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  8. CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site.

    Science.gov (United States)

    Duus, Karen; Thielens, Nicole M; Lacroix, Monique; Tacnet, Pascale; Frachet, Philippe; Holmskov, Uffe; Houen, Gunnar

    2010-12-01

    CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site. This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic material by CD91.

  9. CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site

    DEFF Research Database (Denmark)

    Duus, Karen; Thielens, Nicole M; Lacroix, Monique;

    2010-01-01

    CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found...... to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site....... This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic...

  10. Oxytocin binding sites in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  11. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    Science.gov (United States)

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  12. Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum.

    Science.gov (United States)

    Ran, Yanchao; Thibodeau, Patrick H

    2017-02-03

    ABC transporters are polytopic membrane proteins that utilize ATP binding and hydrolysis to facilitate transport across biological membranes. Forty-eight human ABC transporters have been identified in the genome, and the majority of these are linked to heritable disease. Mutations in the ABCC6 (ATP binding cassette transporter C6) ABC transporter are associated with pseudoxanthoma elasticum, a disease of altered elastic properties in multiple tissues. Although ∼200 mutations have been identified in pseudoxanthoma elasticum patients, the underlying structural defects associated with the majority of these are poorly understood. To evaluate the structural consequences of these missense mutations, a combination of biophysical and cell biological approaches were applied to evaluate the local and global folding and assembly of the ABCC6 protein. Structural and bioinformatic analyses suggested that a cluster of mutations, representing roughly 20% of the patient population with identified missense mutations, are located in the interface between the transmembrane domain and the C-terminal nucleotide binding domain. Biochemical and cell biological analyses demonstrate these mutations influence multiple steps in the biosynthetic pathway, minimally altering local domain structure but adversely impacting ABCC6 assembly and trafficking. The differential impacts on local and global protein structure are consistent with hierarchical folding and assembly of ABCC6. Stabilization of specific domain-domain interactions via targeted amino acid substitution in the catalytic site of the C-terminal nucleotide binding domain restored proper protein trafficking and cell surface localization of multiple biosynthetic mutants. This rescue provides a specific mechanism by which chemical chaperones could be developed for the correction of ABCC6 biosynthetic defects.

  13. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  14. Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

    Science.gov (United States)

    Shrestha, Suresh; Salins, Lyndon L E.; Mark Ensor, C.; Daunert, Sylvia

    2002-01-01

    Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the

  15. Basis for half-site ligand binding in yeast NAD(+)-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Lin, An-Ping; McAlister-Henn, Lee

    2011-09-27

    Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD(+) binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2(C150S) enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD(+) sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1(G15D)/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD(+) site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD(+) binding sites.

  16. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    Institute of Scientific and Technical Information of China (English)

    CHUJin-Shong; WANGYi-Fei

    1989-01-01

    The specific estrogen binding site for 17β-estradiol has been investigated on human spermatozoa by electron microscopec autoradiography. The results show that the binding sites were distributed over the surface of human spermatozoa: acrosomal cap, equatorial

  17. Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli*

    Science.gov (United States)

    Opačić, Milena; Vos, Erwin P. P.; Hesp, Ben H.; Broos, Jaap

    2010-01-01

    The mannitol transporter from Escherichia coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IICmtl). To localize this binding site, 19 single Trp-containing mutants of EIImtl were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IICmtl. Combined with the available two-dimensional projection maps of IICmtl, it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EIImtl becomes phosphorylated at Cys384 in the cytoplasmic B domain. Stably phosphorylated EIImtl mutants were constructed, and FRET experiments showed that the position of mannitol in IICmtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol. PMID:20522557

  18. Localization of the substrate-binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli.

    Science.gov (United States)

    Opacić, Milena; Vos, Erwin P P; Hesp, Ben H; Broos, Jaap

    2010-08-13

    The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.

  19. Being a binding site: characterizing residue composition of binding sites on proteins.

    Science.gov (United States)

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

  20. Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

    Science.gov (United States)

    Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

    2015-01-09

    Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

  1. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...... resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site...

  2. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis.

    Science.gov (United States)

    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki

    2015-12-21

    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans.

  3. Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants

    Energy Technology Data Exchange (ETDEWEB)

    Chigira, Takeru, E-mail: 8120661875@mail.ecc.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nagatoishi, Satoru, E-mail: nagatoishi@bioeng.t.u-tokyo.ac.jp [Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Tsumoto, Kouhei, E-mail: tsumoto@bioeng.t.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2015-08-07

    Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The binding thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα. - Highlights: • Molten globule-likeness of an ERα repressor Prohibitin-2 (PHB2) is identified. • The thermodynamics is validated for the interaction between ERα and PHB2. • PHB2 binds to Y537S and D538G mutants of ERα commonly found in breast cancer. • ERα WT and mutants showed different thermodynamic parameters in the binding to PHB2. • ERα binds to PHB2 with conformational change involving packing of helix 12.

  4. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

    Science.gov (United States)

    Ross, Wilma; Vrentas, Catherine E; Sanchez-Vazquez, Patricia; Gaal, Tamas; Gourse, Richard L

    2013-05-01

    The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

  5. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    Science.gov (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  6. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  7. 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 ...

  8. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  9. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    Energy Technology Data Exchange (ETDEWEB)

    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

  10. Functional identification and characterization of sodium binding sites in Na symporters.

    Science.gov (United States)

    Loo, Donald D F; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A; Wright, Ernest M

    2013-11-19

    Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding.

  11. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    Energy Technology Data Exchange (ETDEWEB)

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike (Vanderbilt)

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  12. Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

    Science.gov (United States)

    Kuno, T; Shuntoh, H; Sakaue, M; Saijoh, K; Takeda, T; Fukuda, K; Tanaka, C

    1988-06-30

    The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

  13. Substrate and drug binding sites in LeuT.

    Science.gov (United States)

    Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan; Marden, Jennifer; Reith, Maarten E; Wang, Da-Neng

    2010-08-01

    LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.

  14. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  15. Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Lin, An-Ping; McAlister-Henn, Lee

    2002-06-21

    Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.

  16. Protein function annotation by local binding site surface similarity.

    Science.gov (United States)

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  17. Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state.

    Science.gov (United States)

    Gu, Jiali; Liu, Min; Guo, Fei; Xie, Wenping; Lu, Wenqiang; Ye, Lidan; Chen, Zhirong; Yuan, Shenfeng; Yu, Hongwei

    2014-02-05

    Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (k(cat)/K(m)) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.

  18. Predicted metal binding sites for phytoremediation

    OpenAIRE

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-01-01

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do no...

  19. Construction and identification of the reporter gene vectors of the 3’UTR region of insulin receptor mRNA including the wild or mutant miR-497 binding site%含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区报告基因载体的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    王美晨; 宁启兰; 李冬民; 王璇; 蓝茜; 李玥; 刘莉; 伊静; 李靖; 宋刘梅; 马莎蕊

    2014-01-01

    目的:利用 pmirGLO Dual-Luciferase miRNA Target Expression Vector(简称为pmirGLO 报告基因载体)构建并鉴定含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR 区报告基因载体(pmir-Insr-3′UTR 及 pmir-mutant-Insr-3′UTR)。方法以大鼠肝脏 cDNA为模板,PCR 获取目的片段(即含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR区);用PmeI、XbaI双酶切 pmirGLO 报告基因载体和含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR区,用T4 DNA连接酶连接纯化后的酶切产物;连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pmir-Insr-3′UTR及 pmir-mutant-Insr-3′UTR重组载体中均插入目的片段;DNA测序结果进一步证实 pmir-Insr-3′UTR重组载体中成功插入了胰岛素受体mRNA 3′UTR区,pmir-mutant-Insr-3′UTR重组载体中成功插入了在miR-497结合位点含有3个突变碱基的胰岛素受体 mRNA 3′UTR 片段。结论成功构建了含 miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR报告基因载体 pmir-Insr-3′UTR及 pmir-mutant-Insr-3′UTR。%Objective To construct and identify the reporter gene vectors of the 3′UTR region of insulin re-ceptor mRNA containing the wild or mutant miR-497 binding site (pmir-Insr-3′UTR and pmir-mutant-Insr-3′UTR) using pmirGLO dual-Luciferase miRNA target expression vector (pmirGLO report gene vector).Methods The target fragments,which were the 3′UTR region of rat insulin receptor mRNA,including wild or mutant miR-497 binding site,were obtained by PCR using rat liver cDNA as template.The target fragments and pmirGLO report gene vector were cut by PmeI and XbaI restriction endonucleases and then the purified enzyme-digested products were ligated together with a T4 DNA ligase.Subsequently,the ligation productions were used to transform DH5αcompetent cells

  20. Structure and localisation of drug binding sites on neurotransmitter transporters.

    Science.gov (United States)

    Ravna, Aina W; Sylte, Ingebrigt; Dahl, Svein G

    2009-10-01

    The dopamine (DAT), serotontin (SERT) and noradrenalin (NET) transporters are molecular targets for different classes of psychotropic drugs. The crystal structure of Aquifex aeolicus LeuT(Aa) was used as a template for molecular modeling of DAT, SERT and NET, and two putative drug binding sites (pocket 1 and 2) in each transporter were identified. Cocaine was docked into binding pocket 1 of DAT, corresponding to the leucine binding site in LeuT(Aa), which involved transmembrane helices (TMHs) 1, 3, 6 and 8. Clomipramine was docked into binding pocket 2 of DAT, involving TMHs 1, 3, 6, 10 and 11, and extracellular loops 4 and 6, corresponding to the clomipramine binding site in a crystal structure of a LeuT(Aa)-clomipramine complex. The structures of the proposed cocaine- and tricyclic antidepressant-binding sites may be of particular interest for the design of novel DAT interacting ligands.

  1. Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

    Energy Technology Data Exchange (ETDEWEB)

    Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.; Davis, Keri D.; Wilson, Wayne A.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med); (Des Moines U)

    2012-12-10

    Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V{sub max}/S{sub 0.5} for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V{sub max}/S{sub 0.5} for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells ({Delta}gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a 'toehold mechanism,' keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

  2. DBD2BS: connecting a DNA-binding protein with its binding sites

    OpenAIRE

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes c...

  3. Identification and characterization of anion binding sites in RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L. (Purdue); (Colorado)

    2010-05-24

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.

  4. The Binding Ability Analysis of the Normal VLDL Receptor and Its Mutant

    Institute of Scientific and Technical Information of China (English)

    QU Shen; FENG Ning; LIU Zhiguo; ZHOU Hua; DENG Yaozu; FENG Zongchen

    2001-01-01

    The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats.To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5.CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeats1-5 were deleted.

  5. Impact of disruption of secondary binding site S2 on dopamine transporter function.

    Science.gov (United States)

    Zhen, Juan; Reith, Maarten E A

    2016-09-01

    The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm.

  6. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1 Mutant Zebrafish.

    Directory of Open Access Journals (Sweden)

    Brian P Grone

    Full Text Available Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1, are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

  7. Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

    Science.gov (United States)

    Gilfoyle, D J; Rodriguez-Lopez, J N; Smith, A T

    1996-03-01

    The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why

  8. Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

    Science.gov (United States)

    Hewitt, Sylvia C; Li, Leping; Grimm, Sara A; Winuthayanon, Wipawee; Hamilton, Katherine J; Pockette, Brianna; Rubel, Cory A; Pedersen, Lars C; Fargo, David; Lanz, Rainer B; DeMayo, Francesco J; Schütz, Günther; Korach, Kenneth S

    2014-06-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

  9. Study of V2 vasopressin receptor hormone binding site using in silico methods.

    Science.gov (United States)

    Sebti, Yeganeh; Sardari, Soroush; Sadeghi, Hamid Mir Mohammad; Ghahremani, Mohammad Hossein; Innamorati, Giulio

    2015-01-01

    The antidiuretic effect of arginine vasopressin (AVP) is mediated by the vasopressin V2 receptor. The docking study of AVP as a ligand to V2 receptor helps in identifying important amino acid residues that might be involved in AVP binding for predicting the lowest free energy state of the protein complex. Whereas previous researchers were not able to detect the exact site of the ligand-receptor binding, we designed the current study to identify the vasopressin V2 receptor hormone binding site using bioinformatic methods. The 3D structure of nonapeptide hormone vasopressin was extracted from Protein Data Bank. Since no suitable template resembling V2 receptor was found, an ab initio approach was chosen to model the protein receptor. Using protein docking methods such as Hex protein-protein docking, the model of V2 receptor was docked to the peptide ligand AVP to identify possible binding sites. The residues that involved in binding site are W293, W296, D297, A300, and P301. The lowest free energy state of the protein complex was predicted after mutation in the above residues. The amount of gained energies permits us to compare the mutant forms with native forms and help to asses critical changes such as positive and negative mutations followed by ranking the best mutations. Based on the mutation/docking predictions, we found some mutants such as W293D and A300E possess positively inducing effect in ligand binding and some of them such as A300R present negatively inducing effect in ligand binding.

  10. A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Kristensen, K D;

    1996-01-01

    with that of the wild-type vector. Thirty-two of 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern of nucleotide differences from the Akv-based vector...... at or around the glutamine tRNA primer binding site. We propose that the forced recombination of primer binding site mutants involves initial priming on endogenous viral sequences and requires template switching during minus-strand synthesis in the region between the neo gene and the mutated primer binding......, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombination partner RNA was found in virions from psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions...

  11. Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants.

    Science.gov (United States)

    Feenstra, K Anton; Starikov, Eugene B; Urlacher, Vlada B; Commandeur, Jan N M; Vermeulen, Nico P E

    2007-03-01

    Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.

  12. Cooperativity between calmodulin-binding sites in Kv7.2 channels.

    Science.gov (United States)

    Alaimo, Alessandro; Alberdi, Araitz; Gomis-Perez, Carolina; Fernández-Orth, Juncal; Gómez-Posada, Juan Camilo; Areso, Pilar; Villarroel, Alvaro

    2013-01-01

    Among the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca(2+) binding protein. Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca(2+), and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.

  13. Molecular modeling and residue interaction network studies on the mechanism of binding and resistance of the HCV NS5B polymerase mutants to VX-222 and ANA598.

    Science.gov (United States)

    Xue, Weiwei; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun

    2014-04-01

    Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about

  14. Influence of sulfhydryl sites on metal binding by bacteria

    Science.gov (United States)

    Nell, Ryan M.; Fein, Jeremy B.

    2017-02-01

    The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5-10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified. In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0 μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low

  15. Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

    Directory of Open Access Journals (Sweden)

    Md Zahid Kamal

    Full Text Available Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

  16. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.;

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable o...

  17. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  18. Differential lipid binding of truncation mutants of Galleria mellonella apolipophorin III.

    Science.gov (United States)

    Dettloff, Matthias; Niere, Marc; Ryan, Robert O; Luty, Robert; Kay, Cyril M; Wiesner, Andreas; Weers, Paul M M

    2002-07-30

    Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3 > H2-4 > H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.

  19. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    Science.gov (United States)

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  20. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    Science.gov (United States)

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  1. SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain (DBD), restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of wild type p53.

    Science.gov (United States)

    Demma, Mark; Maxwell, Eugene; Ramos, Robert; Liang, Lianzhu; Li, Cheng; Hesk, David; Rossman, Randall; Mallams, Alan; Doll, Ronald; Liu, Ming; Seidel-Dugan, Cynthia; Bishop, W Robert; Dasmahapatra, Bimalendu

    2010-04-02

    Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

  2. Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Kramhøft, Birte; Bozonnet, Sophie;

    2012-01-01

    Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380...... is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing...

  3. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Directory of Open Access Journals (Sweden)

    Javier Estrada

    Full Text Available DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  4. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Science.gov (United States)

    Estrada, Javier; Ruiz-Herrero, Teresa; Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  5. Cation binding site of cytochrome c oxidase: progress report.

    Science.gov (United States)

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  6. Vif proteins from diverse primate lentiviral lineages use the same binding site in APOBEC3G.

    Science.gov (United States)

    Letko, Michael; Silvestri, Guido; Hahn, Beatrice H; Bibollet-Ruche, Frederick; Gokcumen, Omer; Simon, Viviana; Ooms, Marcel

    2013-11-01

    APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty mangabey monkeys (SIVsmm), suggesting that the A3G binding site for Vif proteins of the SIVsmm/HIV-2 lineage differs from that of HIV-1. To map the SIVsmm Vif binding site of A3G, we performed immunoprecipitations of individual A3G domains, Vif/A3G degradation assays and a detailed mutational analysis of human A3G. We show that A3G residue 129, but not the adjacent position 128, confers susceptibility to degradation by SIVsmm Vif. An artificial A3G mutant, the P129D mutant, was resistant to degradation by diverse Vifs from HIV-1, HIV-2, SIVagm, and chimpanzee SIV (SIVcpz), suggesting a conserved lentiviral Vif binding site. Gorilla A3G naturally contains a glutamine (Q) at position 129, which makes its A3G resistant to Vifs from diverse lineages. We speculate that gorilla A3G serves as a barrier against SIVcpz strains. In summary, we show that Vif proteins from distinct lineages bind to the same A3G loop, which includes positions 128 and 129. The multiple adaptations within this loop among diverse primates underscore the importance of counteracting A3G in lentiviral evolution.

  7. Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb5 and Its Mutants by Electrospray Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    YU,Chong-Tian(余翀天); GUO,Yin-Long(郭寅龙); L(U),Long(吕龙); WANG,Yun-Hua(王韵华); YAO,Ping(姚萍); HUANG,Zhong-Xian(黄仲贤)

    2002-01-01

    To ehucidate the effect of various amino acid residues on the heme-binding pocket in cytochrome Tbs, several residues were chosen for replacement by means of site-directed mutagenesis.Comparison of the mass spectrmn between the F35Y mutant and the wild type shows that the relative abundance of holoprotein ion of F35Y is lower than that of the wild type in gas phase. It is concluded that mutation from Phe35 residue to tyrosine decreases the hydrophobic character of cytochrome Tbs heme pocket, which decreases the stability of heme-binding pocket. ESI-MS spectra of the mutants V61E, V61K, V61H and V61Y show various contribution of amino acid to the stability of heme-binding pocket. The small and non-polar residue Vat61 was replaced with large or polar residues, resulting in enhancing the trend of heme leaving from the pocket. In addition, comparison of the mass relative abundance of bolo-proteins among all the Va161-mutants, shows that their stability in gas phase appropriately submit the following order: wild type > V61H > V61E > V61K ≈ V61Y. The extra great stability of quadruple sites mutant E44/48/56A/D60A shows that reduction of electrostatic or hydrogen bond interactions among the residues locating in the outside region of the heme edge remarkably affect the stability of heme. The results of analyzing the oxidation states of heme iron in Tbs and its mutants by insource-CAD experiment suggest that the charge states of heme iron maintain inflexible in mutation process.

  8. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

    Directory of Open Access Journals (Sweden)

    E. Gout

    2010-01-01

    Full Text Available Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

  9. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  10. Differential effects of methoxy group on the interaction of curcuminoids with two major ligand binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Hiroki Sato

    Full Text Available Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA clearly indicated that curcumin (Cur, demethylcurcumin (Dmc and bisdemethoxycurcumin (Bdmc bind to both Site I (sub-site Ia and Ib and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.

  11. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  12. Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.

    Science.gov (United States)

    Li, Z; Deber, C M

    1991-10-31

    Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.

  13. Modeling lanthanide series binding sites on humic acid.

    Science.gov (United States)

    Pourret, Olivier; Martinez, Raul E

    2009-02-01

    Lanthanide (Ln) binding to humic acid (HA) has been investigated by combining ultrafiltration and ICP-MS techniques. A Langmuir-sorption-isotherm metal-complexation model was used in conjunction with a linear programming method (LPM) to fit experimental data representing various experimental conditions both in HA/Ln ratio (varying between 5 and 20) and in pH range (from 2 to 10) with an ionic strength of 10(-3) mol L(-1). The LPM approach, not requiring prior knowledge of surface complexation parameters, was used to solve the existing discrepancies in LnHA binding constants and site densities. The application of the LPM to experimental data revealed the presence of two discrete metal binding sites at low humic acid concentrations (5 mg L(-1)), with log metal complexation constants (logK(S,j)) of 2.65+/-0.05 and 7.00 (depending on Ln). The corresponding site densities were 2.71+/-0.57x10(-8) and 0.58+/-0.32x10(-8) mol of Ln(3+)/mg of HA (depending on Ln). Total site densities of 3.28+/-0.28x10(-8), 4.99+/-0.02x10(-8), and 5.01+/-0.01x10(-8) mol mg(-1) were obtained by LPM for humic acid, for humic acid concentrations of 5, 10, and 20 mg L(-1), respectively. These results confirm that lanthanide binding occurs mainly at weak sites (i.e., ca. 80%) and second at strong sites (i.e., ca. 20%). The first group of discrete metal binding sites may be attributed to carboxylic groups (known to be the main binding sites of Ln in HA), and the second metal binding group to phenolic moieties. Moreover, this study evidences heterogeneity in the distribution of the binding sites among Ln. Eventually, the LPM approach produced feasible and reasonable results, but it was less sensitive to error and did not require an a priori assumption of the number and concentration of binding sites.

  14. The Human p73 Promoter: Characterization and Identification of Functional E2F Binding Sites

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    Ratnam S. Seelan

    2002-01-01

    Full Text Available p73, a member of the p53 family, is overexpressed in many cancers. To understand the mechanism(s underlying this overexpression, we have undertaken a detailed characterization of the human p73 promoter. The promoter is strongly activated in cells expressing exogenous E2F1 and suppressed by exogenous Rb. At least three functional E2F binding sites, located immediately upstream of exon 1 (at-284,-155 and-132 mediate this induction. 5' serially deleted promoter constructs and constructs harboring mutated E2F sites were analyzed for their response to exogenously expressed E2F1 or Rb to establish functionality of these sites. Authenticity of E2F sites was further confirmed by electrophoretic mobility shift assay (EMSA using E2F1 /DP1 heterodimers synthesized in vitro, followed by competition assays with unlabeled wild-type or mutant oligonucleotides and supershift analysis using anti-E2F1 antibodies. In vivo binding of E2F1 to the p73 promoter was demonstrated using nuclear extracts prepared from E2F1-inducible Saos2 cells. The region conferring the highest promoter activity was found to reside between-113 to-217 of the p73 gene. Two of the three functional E2F sites (at-155 and-132 reside within this region. Our results suggest that regulation of p73 expression is primarily mediated through binding of E2 F1 to target sites at-155 and-132.

  15. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  16. Probing binding hot spots at protein-RNA recognition sites.

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity.

  17. Probing binding hot spots at protein–RNA recognition sites

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-01

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein–RNA interfaces to probe the binding hot spots at protein–RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein–protein and protein–RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein–RNA recognition sites with desired affinity. PMID:26365245

  18. Identification of the molecular site of ivabradine binding to HCN4 channels.

    Directory of Open Access Journals (Sweden)

    Annalisa Bucchi

    Full Text Available Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the "open channel block", the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.

  19. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    Energy Technology Data Exchange (ETDEWEB)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-27

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics.

  20. Autoradiographic localization of estrogen binding sites in human mammary lesions

    Energy Technology Data Exchange (ETDEWEB)

    Buell, R.H.

    1984-01-01

    The biochemical assay of human mammary carcinomas for estrogen receptors is of proven clinical utility, but the cellular localization of estrogen binding sites within these lesions is less certain. The author describes the identification of estrogen binding sites as visualized by thaw-mount autoradiography after in vitro incubation in a series of 17 benign and 40 malignant human female mammary lesions. The results on the in vitro incubation method compared favorably with data from in vivo studies in mouse uterus, a well-characterized estrogen target organ. In noncancerous breast biopsies, a variable proportion of epithelial cells contained specific estrogen binding sites. Histologically identifiable myoepithelial and stromal cells were, in general, unlabeled. In human mammary carcinomas, biochemically estrogen receptor-positive, labeled and unlabeled neoplastic epithelial cells were identified by autoradiography. Quantitative results from the autoradiographic method compared favorably with biochemical data.

  1. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  2. A novel non-opioid binding site for endomorphin-1.

    Science.gov (United States)

    Lengyel, I; Toth, F; Biyashev, D; Szatmari, I; Monory, K; Tomboly, C; Toth, G; Benyhe, S; Borsodi, A

    2016-08-01

    Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the μ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the μ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the μ-opioid receptor. Our recent data indicate that a radiolabelled [(3)H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems

  3. Hydrophobicity of reactive site loop of SCCA1 affects its binding to hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Min Chen; Tong Cheng; Chen-Yu Xu; Ting Wu; Shan-Hai Ou; Tao Zhang; Jun Zhang; Ning-Shao Xia

    2005-01-01

    AIM: To investigate the role of SCCA2 and other SCCA1 molecules in the process of hepatitis B virus (HBV) binding to mammalian cells.METHODS: SCCA1 and SCCA2 were isolated from HepG2. Binding protein (BP) genes were obtained through PCR. Recombinant baculoviruses expressing SCCA1, SCCA2, BP, and different mutants were constructed and utilized to infect mammalian cells to investigate the binding ability of infected cells to HBV.RESULTS: A SCCA1 gene (A1) was isolated from HepG2, but it appeared to lack the binding ability of infected cells to HBV. Two mutants, A1-BP and BP-A1, were constructed by interchanging the carboxyl terminal of A1 and BP. Cells expressing A1-BP showed an increased virus bindingcapacity, but not BP-A1. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them were found in the reactive site loop (RSL) of SCCA1. Primary structure assay revealed that the hydrophobicity of BP and AJ515706 in this domain was strong, but A1 was relatively weak. Changing the aa349 of A1 from low hydrophobic glutamic acid to high hydrophobic valine enhanced HBV binding. In contrast, HBV binding was reduced by changing the aa349 of BP from valine to glutamic acid. CONCLUSION: The reslts suggest that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.

  4. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    Niu Huang

    Full Text Available The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

  5. Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

    Directory of Open Access Journals (Sweden)

    Yibo Jiang

    2013-12-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM, a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1–119 from the pSN and cSN (cattle sialoadhesin N-termini (excluding the 19-amino acid signal peptide were modeled via homology modeling based on mSN (mouse sialoadhesin template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting, FAR-WB (far Western blotting, ELISA (enzyme-linked immunosorbent assay and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies.

  6. Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides

    Institute of Scientific and Technical Information of China (English)

    肖谷田; 谢毅; 张婕; 孙筱清; 吴小舟; 陈小央; 王启松

    1997-01-01

    A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.

  7. Characterization of Heparin-binding Site of Tissue Transglutaminase

    Science.gov (United States)

    Wang, Zhuo; Collighan, Russell J.; Pytel, Kamila; Rathbone, Daniel L.; Li, Xiaoling; Griffin, Martin

    2012-01-01

    Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. PMID:22298777

  8. Eel calcitonin binding site distribution and antinociceptive activity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  9. Location of the coenzyme binding site in the porcine mitochondrial NADP-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Huang, Yu Chu; Colman, Roberta F

    2005-08-26

    The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

  10. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    Science.gov (United States)

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  11. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Science.gov (United States)

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  12. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  13. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

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

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  14. Incorporating evolution of transcription factor binding sites into annotated alignments

    Indian Academy of Sciences (India)

    Abha S Bais; Steffen Grossmann; Martin Vingron

    2007-08-01

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield ``conserved TFBSs”. Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair-profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions, as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs, we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification

  15. The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases.

    Science.gov (United States)

    Pagh, Rasmus; Duus, Karen; Laursen, Inga; Hansen, Paul R; Mangor, Julie; Thielens, Nicole; Arlaud, Gérard J; Kongerslev, Leif; Højrup, Peter; Houen, Gunnar

    2008-02-01

    The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.

  16. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  17. Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

    Science.gov (United States)

    Armitage, Ian M; Drakenberg, Torbjörn; Reilly, Brian

    2013-01-01

    Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd

  18. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  19. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  20. Computational studies on the resistance of penicillin-binding protein 2B (PBP2B) of wild-type and mutant strains of Streptococcus pneumoniae against β-lactam antibiotics.

    Science.gov (United States)

    Ramalingam, Jothi; Vennila, Jannet; Subbiah, Parthasarathy

    2013-09-01

    Mutations within transpeptidase domain of penicillin-binding protein 2B of the strains of Streptococcus pneumoniae leads to resistance against β-lactam antibiotics. To uncover the important residues responsible for sensitivity and resistance, the recently determined three dimensional structures of penicillin-binding protein 2B of both wild-type R6 (sensitive) and mutant 5204 (resistant) strains along with the predicted structures of other mutant strains G54, Hungary19A-6 and SP195 were considered for the interaction study with β-lactam antibiotics using induced-fit docking of Schrödinger. Associated binding energies of the complexes and their intermolecular interactions in the binding site clearly show that the wild-type R6 as sensitive, mutant strains 5204 and G54 as highly resistant, and the mutant strains Hungary19A-6 and SP195 as intermediate resistant. The study also reveals that the mutant strains Hungary19A-6 and SP195 exhibit intermediate resistant because of the existence of mutations till the intermediate 538th and 516th positions, respectively, and not till the end of the C-terminus. Furthermore, our investigations show that if the mutations are extended till the end of the C terminus, then the antibiotic resistance of induced-mutated strains increases from intermediate to high as in the strains 5204 and G54. The binding patterns obtained in the study are useful in designing potential inhibitors against multidrug resistant S. pneumoniae.

  1. Copper(II) and nickel(II) binding sites of peptide containing adjacent histidyl residues.

    Science.gov (United States)

    Grenács, Ágnes; Sanna, Daniele; Sóvágó, Imre

    2015-10-01

    Copper(II) and nickel(II) complexes of the terminally protected nonapeptide Ac-SGAEGHHQK-NH2 modeling the metal binding sites of the (8-16) domain of amyloid-β have been studied by potentiometric, UV-vis, CD and ESR spectroscopic methods. The studies on the mutants containing only one of the histidyl residues (Ac-SGAEGAHQK-NH2, Ac-SGAEGHAQK-NH2) have also been performed. The formation of imidazole and amide coordinated mononuclear complexes is characteristic of all systems with a preference of nickel(II) binding to the His14 site, while the involvement of both histidines in metal binding is suggested in the corresponding copper(II) complexes. The formation of bis(ligand) and dinuclear complexes has also been observed in the copper(II)-Ac-SGAEGHHQK-NH2 system. The results provide further support for the copper(II) binding ability of the (8-16) domain of amyloid-β and support the previous assumptions that via the bis(ligand) complex formation copper(II) ions may promote the formation of the oligomers of amyloid-β.

  2. Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

    Science.gov (United States)

    Bakhautdin, Bakytzhan; Goksoy Bakhautdin, Esen; Fox, Paul L

    2014-10-31

    Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

  3. Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

    Energy Technology Data Exchange (ETDEWEB)

    Buhrman, Greg; O; #8242; Connor, Casey; Zerbe, Brandon; Kearney, Bradley M.; Napoleon, Raeanne; Kovrigina, Elizaveta A.; Vajda, Sandor; Kozakov, Dima; Kovrigin, Evgenii L.; Mattos, Carla (NCSU); (MCW); (BU)

    2012-09-17

    We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

  4. (/sup 3/H)desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons

    Energy Technology Data Exchange (ETDEWEB)

    Baeckstroem, I.T.Ro.; Ross, S.B.; Marcusson, J.O.

    1989-04-01

    The pharmacological and biochemical characteristics of (3H)desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one (3H)desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The (3H)desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 microM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of approximately 1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). In contrast, when 10 microM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with KD values of 0.5 and greater than 100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 microM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of (3H)noradrenaline uptake but not with 5-(3H)hydroxytryptamine uptake.

  5. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Science.gov (United States)

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste.

  6. Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Yates Robert B

    2002-02-01

    Full Text Available Abstract Background The metallo-β-lactamases are Zn(II-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. Results Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. Conclusions The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases.

  7. Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

    Science.gov (United States)

    Swoboda, Ines; De Weerd, Nicole; Bhalla, Prem L; Niederberger, Verena; Sperr, W R; Valent, Peter; Kahlert, Helga; Fiebig, Helmut; Verdino, Petra; Keller, Walter; Ebner, Christof; Spitzauer, Susanne; Valenta, Rudolf; Singh, Mohan B

    2002-01-01

    More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

  8. The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase.

    Science.gov (United States)

    Francia, Francesco; Giachini, Lisa; Boscherini, Federico; Venturoli, Giovanni; Capitanio, Giuseppe; Martino, Pietro Luca; Papa, Sergio

    2007-02-20

    EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn(2+) with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O-COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.

  9. Minimal Zn2+ Binding Site of Amyloid-β

    Science.gov (United States)

    Tsvetkov, Philipp O.; Kulikova, Alexandra A.; Golovin, Andrey V.; Tkachev, Yaroslav V.; Archakov, Alexander I.; Kozin, Sergey A.; Makarov, Alexander A.

    2010-01-01

    Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6–14 as the minimal Zn2+ binding site wherein the ion is coordinated by His6, Glu11, His13, and His14. With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11–14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn2+-induced aggregation of Aβ. PMID:21081056

  10. Minimal Zn(2+) binding site of amyloid-β.

    Science.gov (United States)

    Tsvetkov, Philipp O; Kulikova, Alexandra A; Golovin, Andrey V; Tkachev, Yaroslav V; Archakov, Alexander I; Kozin, Sergey A; Makarov, Alexander A

    2010-11-17

    Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aβ.

  11. Evolutionary computation for discovery of composite transcription factor binding sites

    OpenAIRE

    Fogel, Gary B.; Porto, V. William; Varga, Gabor; Dow, Ernst R.; Craven, Andrew M.; Powers, David M.; Harlow, Harry B.; Su, Eric W.; Onyia, Jude E.; Su, Chen

    2008-01-01

    Previous research demonstrated the use of evolutionary computation for the discovery of transcription factor binding sites (TFBS) in promoter regions upstream of coexpressed genes. However, it remained unclear whether or not composite TFBS elements, commonly found in higher organisms where two or more TFBSs form functional complexes, could also be identified by using this approach. Here, we present an important refinement of our previous algorithm and test the identification of composite elem...

  12. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    Full Text Available BACKGROUND: The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates. METHODOLOGY/PRINCIPAL FINDINGS: We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data. CONCLUSIONS/SIGNIFICANCE: Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct

  13. Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

    Energy Technology Data Exchange (ETDEWEB)

    Tejedor, F.; Amils, R.; Ballesta, J.P.

    1985-07-02

    Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of (/sup 125/I)iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.

  14. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  15. Cloud Computing for Protein-Ligand Binding Site Comparison

    Directory of Open Access Journals (Sweden)

    Che-Lun Hung

    2013-01-01

    Full Text Available The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  16. Creation of a putative third metal binding site in type II dihydroorotases significantly enhances enzyme activity.

    Science.gov (United States)

    Huang, Yen-Hua; Huang, Cheng-Yang

    2015-01-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.

  17. Direct GR Binding Sites Potentiate Clusters of TF Binding across the Human Genome.

    Science.gov (United States)

    Vockley, Christopher M; D'Ippolito, Anthony M; McDowell, Ian C; Majoros, William H; Safi, Alexias; Song, Lingyun; Crawford, Gregory E; Reddy, Timothy E

    2016-08-25

    The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs.

  18. Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Senior, Alan E

    2004-07-23

    Residues responsible for phosphate binding in F(1)F(0)-ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved betaArg-246 in a suitable position to bind phosphate. Mutations betaR246Q, betaR246K, and betaR246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F(1) by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by approximately 50%, although reaction still occurred at residue betaTyr-297, proximal to betaArg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the betaE catalytic site of E. coli F(1) and that betaArg-246 is an important phosphate-binding residue.

  19. Upstream CRP-binding site is not essential for CRP-cAMP-mediated inhibition on the nifU promoter

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    When the NifA-mediated activation of Klebsiella pneumoniae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified NifA-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter. These results suggest that direct interaction between CRP and Eσ54, other than the DNA binding site(s) competition between CRP and NifA, plays the principal role in the CRP-cAMP-mediated inhibitory effect on nifU.

  20. Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation.

    Science.gov (United States)

    Shima, F; Okada, T; Kido, M; Sen, H; Tanaka, Y; Tamada, M; Hu, C D; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    2000-01-01

    Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.

  1. Steered molecular dynamics study of inhibitor binding in the internal binding site in dehaloperoxidase-hemoglobin.

    Science.gov (United States)

    Zhang, Zhisen; Santos, Andrew P; Zhou, Qing; Liang, Lijun; Wang, Qi; Wu, Tao; Franzen, Stefan

    2016-04-01

    The binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm(2)) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet-visible (UV-vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.

  2. Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site.

    Science.gov (United States)

    Naik, Pradeep K; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C

    2011-06-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

  3. Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site

    DEFF Research Database (Denmark)

    Maric, Hans-Michael; Mukherjee, Jayanta; Tretter, Verena

    2011-01-01

    confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues...... that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key...

  4. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Directory of Open Access Journals (Sweden)

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  5. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Science.gov (United States)

    Martinez-Archundia, Marlet; Cordomi, Arnau; Garriga, Pere; Perez, Juan J.

    2012-01-01

    The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand. PMID:22500107

  6. Molecular modeling and competition binding study of Br-noscapine and colchicine provides insight into noscapinoid-tubulin binding site

    OpenAIRE

    Naik, Pradeep K.; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C.

    2011-01-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: 1) In silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; 2) the molecular mechanics-generalized Born/surface area (MM-GB/SA)...

  7. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    Science.gov (United States)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  8. Characterization of Binding Sites of Eukaryotic Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    Jiang Qian; Jimmy Lin; Donald J. Zack

    2006-01-01

    To explore the nature of eukaryotic transcription factor (TF) binding sites and determine how they differ from surrounding DNA sequences, we examined four features associated with DNA binding sites: G+C content, pattern complexity,palindromic structure, and Markov sequence ordering. Our analysis of the regulatory motifs obtained from the TRANSFAC database, using yeast intergenic sequences as background, revealed that these four features show variable enrichment in motif sequences. For example, motif sequences were more likely to have palindromic structure than were background sequences. In addition, these features were tightly localized to the regulatory motifs, indicating that they are a property of the motif sequences themselves and are not shared by the general promoter "environment" in which the regulatory motifs reside. By breaking down the motif sequences according to the TF classes to which they bind, more specific associations were identified. Finally, we found that some correlations, such as G+C content enrichment, were species-specific, while others, such as complexity enrichment, were universal across the species examined. The quantitative analysis provided here should increase our understanding of protein-DNA interactions and also help facilitate the discovery of regulatory motifs through bioinformatics.

  9. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  10. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Vedula, L. Sangeetha; Brannigan, Grace; Economou, Nicoleta J.; Xi, Jin; Hall, Michael A.; Liu, Renyu; Rossi, Matthew J.; Dailey, William P.; Grasty, Kimberly C.; Klein, Michael L.; Eckenhoff, Roderic G.; Loll, Patrick J.; (Drexel-MED); (UPENN)

    2009-10-21

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  11. A Unitary Anesthetic-Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Vedula, L.; Brannigan, G; Economou, N; Xi, J; Hall, M; Liu, R; Rossi, M; Dailey, W; Grasty, K; et. al.

    2009-01-01

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  12. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    L Vedula; G Brannigan; N Economou; J Xi; M Hall; R Liu; M Rossi; W Dailey; K Grasty; et. al.

    2011-12-31

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  13. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  14. Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

    Science.gov (United States)

    Wang, J; Suzuki, N; Nishida, Y; Kataoka, T

    1993-07-01

    In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

  15. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher;

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a cat......Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  16. Organizational requirements of the SaeR binding sites for a functional P1 promoter of the sae operon in Staphylococcus aureus.

    Science.gov (United States)

    Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; Bae, Taeok

    2012-06-01

    In Staphylococcus aureus, the SaeRS two-component system controls the expression of multiple virulence factors. Of the two promoters in the sae operon, P1 is autoinduced and has two binding sites for the response regulator SaeR. In this study, we examined the organizational requirements of the SaeR binding sites in P1 for transcription activation. Mutational studies showed that both binding sites are essential for binding to phosphorylated SaeR (P-SaeR) and transcription activation. When the 21-bp distance between the centers of the two SaeR binding sites was altered to 26 bp, 31 bp, 36 bp, or 41 bp, only the 31-bp mutant retained approximately 40% of the original promoter activity. When the -1-bp spacing (i.e.,1-bp overlap) between the primary SaeR binding site and the -35 promoter region was altered, all mutant P1 promoters failed to initiate transcription; however, when the first nucleotide of the -35 region was changed from A to T, the mutants with 0-bp or 22-bp spacing showed detectable promoter activity. Although P-SaeR was essential for the binding of RNA polymerase to P1, it was not essential for the binding of the enzyme to the alpha-hemolysin promoter. When the nonoptimal spacing between promoter elements in P1 or the coagulase promoter was altered to the optimal spacing of 17 bp, both promoters failed to initiate transcription. These results suggest that SaeR binding sites are under rather strict organizational restrictions and provide clues for understanding the molecular mechanism of sae-mediated transcription activation.

  17. Crystallographic Location and Mutational Analysis of Zn and Cd Inhibitory Sites and Role of Lipidic Carboxylates in Rescuing Proton Path Mutants in Cytochrome c Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Mills, Denise A.; Hiser, Carrie; Murphree, Anna; Garavito, R. Michael; Ferguson-Miller, Shelagh; Hosler, Jonathan (MSU); (UMMC)

    2009-01-15

    Cytochrome c oxidase (CcO) transfers protons from the inner surface of the enzyme to the buried O{sub 2} reduction site through two different pathways, termed K and D, and from the outer surface via an undefined route. These proton paths can be inhibited by metals such as zinc or cadmium, but the sites of inhibition have not been established. Anomalous difference Fourier analyses of Rhodobacter sphaeroides CcO crystals, with cadmium added, reveal metal binding sites that include the proposed initial proton donor/acceptor of the K pathway, Glu-101 of subunit II. Mutant forms of CcO that lack Glu-101{sub II} (E101A and E101A/H96A) exhibit low activity and eliminate metal binding at this site. Significant activity is restored to E101A and E101A/H96A by adding the lipophilic carboxylic compounds, arachidonic acid and cholic acid, but not by their non-carboxylic analogues. These amphipathic acids likely provide their carboxylic groups as substitute proton donors/acceptors in the absence of Glu-101{sub II}, as previously observed for arachidonic acid in mutants that alter Asp-132{sub I} of the D pathway. The activity of E101A/H96A is still inhibited by zinc, but this remaining inhibition is nearly eliminated by removal of subunit III, which is known to alter the D pathway. The results identify the Glu-101/His-96 site of subunit II as the site of metal binding that inhibits the uptake of protons into the K pathway and indicate that subunit III contributes to zinc binding and/or inhibition of the D pathway. By removing subunit III from E101A/H96A, thereby eliminating zinc inhibition of the uptake of protons from the inner surface of CcO, we confirm that an external zinc binding site is involved in inhibiting the backflow of protons to the active site.

  18. A new F131V mutation in Chlamydomonas phytoene desaturase locates a cluster of norflurazon resistance mutations near the FAD-binding site in 3D protein models.

    Directory of Open Access Journals (Sweden)

    Julio V Suarez

    Full Text Available The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM. The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505 but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene.

  19. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    CERN Document Server

    Clifford, Jacob

    2015-01-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through Position Weight Matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain a...

  20. Cytosine hypomethylation at CHG and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus roseus

    Indian Academy of Sciences (India)

    Renu Kumari; Gitanjali Yadav; Vishakha Sharma; Vinay Sharma; Sushil Kumar

    2013-12-01

    The 5S and 18S rDNA sequences of Catharanthus roseus cv ‘Nirmal’ (wild type) and its leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill) single mutants and lli egd, lli ill and egd ill double mutants were characterized. The lli, egd and ill mutants of Mendelian inheritance bore the names after their most conspicuous morphological feature(s). They had been chemically induced and isolated for their salt tolerance. The double mutants were isolated as morphological segregants from crosses between single mutants. The morphological features of the two parents accompanied salt tolerance in the double mutants. All the six mutants were hypomethylated at repeat sequences, upregulated and downregulated for many genes and carried pleiotropic alterations for several traits. Here the 5S and 18S rDNAs of C. roseus were found to be relatively low in cytosine content. Cytosines were preponderantly in CG context (53%) and almost all of them were methylated (97%). The cytosines in CHH and CHG (where H = A, T or C) contexts were largely demethylated (92%) in mutants. The demethylation was attributable to reduced expression of RDR2 and DRM2 led RNA dependant DNA methylation and CMT3 led maintenance methylation pathways. Mutants had gained some cytosines by substitution of C at T sites. These perhaps arose on account of errors in DNA replication, mediated by widespread cytosine demethylation at CHG and CHH sites. It was concluded that the regulation of cytosine methylation mechanisms was disturbed in the mutants. ILL, EGD and LLI genes were identified as the positive regulators of other genes mediating the RdDM and CMT3 pathways, for establishment and maintenance of cytosine methylation in C. roseus.

  1. CtBP1/BARS Gly172-->Glu mutant structure: impairing NAD(H)-binding and dimerization.

    Science.gov (United States)

    Nardini, Marco; Valente, Carmen; Ricagno, Stefano; Luini, Alberto; Corda, Daniela; Bolognesi, Martino

    2009-03-27

    C-terminal binding proteins (CtBPs) are multi-functional proteins involved in nuclear transcriptional co-repression, Golgi membrane fission, and synaptic ribbon formation. Binding of NAD(H) to CtBPs promotes dimerization. CtBP dimers act as a scaffold for multimeric protein complex formation, thus bridging transcriptional repressors and their targets in the nucleus. Based on size-exclusion chromatography experiments and on the crystal structure of the NAD(H)-free G172E CtBP mutant, we show here that absence of NAD(H) induces flexibility/backbone conformational changes at the dimerization interface and at the CtBP interdomain region. The results presented shed first light on the correlation between NAD(H)-binding and functional CtBP dimerization.

  2. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  3. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  4. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi (Osaka Univ. (Japan)); Takenaka, Hitoshi; Hamada, Minoru (Miyazaki Medical College (Japan)); Kuby, S.A. (Univ. of Utah, Salt Lake City (USA))

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP{sup 2{minus}} and for AMP{sup 2{minus}} in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP{sup 2{minus}} and MgATP{sup 2{minus}}. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K{sub m,app} values for AMP{sup 2{minus}} of the mutant enzymes, the relatively small increases in the K{sub m,app} values for MgATP{sup 2{minus}}, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site.

  5. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  6. DBD2BS: connecting a DNA-binding protein with its binding sites.

    Science.gov (United States)

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-07-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein-DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD-DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein-DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

  7. Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair

    DEFF Research Database (Denmark)

    Dherin, Claudine; Gueneau, Emeric; Francin, Mathilde;

    2009-01-01

    , and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting...... protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2...

  8. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  9. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.;

    2006-01-01

    as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile...

  10. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  11. Monoclonal Anti—CD4 Antibody MT310 Binds HIV-1 gp120 Binding Site on CD4

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Tests show the monoclonal anti—CD4 antibody (mAb) MT310 recognizes the gp120-binding site on CD4 as part of its mechanism for strongly inhibiting human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells. In competition tests, mAb MT310 and mAb Leu3a (an anti-CD4 mAb recognizing the gp120-binding site) all inhibited gp120-binding to CD4+ T lymphocytes, while mAb MT405 did not. This result suggests that MT310, like Leu3a, recognizes the gp120-binding site on CD4. To further confirm whether MT310 recognizes the gp120-binding site on CD4, we prepared rabbit anti-idiotypic antisera (Ab2) against MT310 (Ab1). The anti-idiotypic antisera against MT310 inhibited binding of MT310 and Leu3a to human CD4+ T lymphocytes, but did not block binding of MT151 with the second domain of CD4, while rabbit anti-idiotypic antisera to MT151 could block binding of itself to these cells, but could not inhibit the binding of MT310 and Leu3a, further indicating that MT310 recognized the gp120-binding site on CD4.

  12. ATLAS: A database linking binding affinities with structures for wild-type and mutant TCR-pMHC complexes.

    Science.gov (United States)

    Borrman, Tyler; Cimons, Jennifer; Cosiano, Michael; Purcaro, Michael; Pierce, Brian G; Baker, Brian M; Weng, Zhiping

    2017-02-03

    The ATLAS (Altered TCR Ligand Affinities and Structures) database (https://zlab.umassmed.edu/atlas/web/) is a manually curated repository containing the binding affinities for wild-type and mutant T cell receptors (TCRs) and their antigens, peptides presented by the major histocompatibility complex (pMHC). The database links experimentally measured binding affinities with the corresponding three dimensional (3D) structures for TCR-pMHC complexes. The user can browse and search affinities, structures, and experimental details for TCRs, peptides, and MHCs of interest. We expect this database to facilitate the development of next-generation protein design algorithms targeting TCR-pMHC interactions. ATLAS can be easily parsed using modeling software that builds protein structures for training and testing. As an example, we provide structural models for all mutant TCRs in ATLAS, built using the Rosetta program. Utilizing these structures, we report a correlation of 0.63 between experimentally measured changes in binding energies and our predicted changes. Proteins 2017. © 2017 Wiley Periodicals, Inc.

  13. Function of IHF in lambda DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB.

    Science.gov (United States)

    Xin, W; Feiss, M

    1993-03-20

    Integration host factor (IHF) plays an accessory role in lambda DNA packaging. IHF affects the interaction of the lambda DNA packaging protein, terminase, with cos, the site on lambda DNA at which terminase binds and introduces staggered nicks to generate cohesive ends of mature lambda chromosomes. cos includes cosB, the terminase binding site and cosN, the adjacent nicking site. cosB includes multiple binding sites for gpNu1, the small subunit of terminase, and an IHF binding site, I1. I1 contains two overlapping sequences, called I1A and I1B, that closely match the consensus sequence for IHF binding sites. The I1A sequence was determined to be the site of IHF binding by hydroxyl radical footprinting experiments. Comparison of the pattern of IHF-induced enhancements and diminishments at I1 with published patterns for IHF binding sites at the lambda attachment site identifies I1A as the IHF binding site at I1. The conclusion that I1A is the IHF binding site was confirmed by studies with DNA mutant in I1A. The I1A- mutation, consisting of three adjacent base-pair changes in I1A, abolished IHF binding. In contrast to the I1A- mutation, a mutation in I1B, also consisting of three adjacent base-pair changes, caused a reduction in the affinity of IHF for I1A, and caused a reduction in the magnitude of the net intrinsic bending of cos lambda.

  14. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  15. Leveraging cross-species transcription factor binding site patterns

    DEFF Research Database (Denmark)

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward;

    2014-01-01

    diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele......Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central...... to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2...

  16. Genome-wide prediction, display and refinement of binding sites with information theory-based models

    Directory of Open Access Journals (Sweden)

    Leeder J Steven

    2003-09-01

    Full Text Available Abstract Background We present Delila-genome, a software system for identification, visualization and analysis of protein binding sites in complete genome sequences. Binding sites are predicted by scanning genomic sequences with information theory-based (or user-defined weight matrices. Matrices are refined by adding experimentally-defined binding sites to published binding sites. Delila-Genome was used to examine the accuracy of individual information contents of binding sites detected with refined matrices as a measure of the strengths of the corresponding protein-nucleic acid interactions. The software can then be used to predict novel sites by rescanning the genome with the refined matrices. Results Parameters for genome scans are entered using a Java-based GUI interface and backend scripts in Perl. Multi-processor CPU load-sharing minimized the average response time for scans of different chromosomes. Scans of human genome assemblies required 4–6 hours for transcription factor binding sites and 10–19 hours for splice sites, respectively, on 24- and 3-node Mosix and Beowulf clusters. Individual binding sites are displayed either as high-resolution sequence walkers or in low-resolution custom tracks in the UCSC genome browser. For large datasets, we applied a data reduction strategy that limited displays of binding sites exceeding a threshold information content to specific chromosomal regions within or adjacent to genes. An HTML document is produced listing binding sites ranked by binding site strength or chromosomal location hyperlinked to the UCSC custom track, other annotation databases and binding site sequences. Post-genome scan tools parse binding site annotations of selected chromosome intervals and compare the results of genome scans using different weight matrices. Comparisons of multiple genome scans can display binding sites that are unique to each scan and identify sites with significantly altered binding strengths

  17. Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1.

    Science.gov (United States)

    Pham, Thu-Hang; Minderjahn, Julia; Schmidl, Christian; Hoffmeister, Helen; Schmidhofer, Sandra; Chen, Wei; Längst, Gernot; Benner, Christopher; Rehli, Michael

    2013-07-01

    The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.

  18. Distribution of intercalative dye binding sites in chromatin.

    Science.gov (United States)

    Lurquin, P F; Seligy, V L

    1976-04-01

    Actinomycin D (AMD) and ethidium bromide (EB) were found to bind to chromatin isolated from a variety of gander tissues according to a strong and weak process analogous to that found for deproteinized DNA. Distribution of the dye intercalation sites in chromatin and DNA were evaluated at low r-values (dye bound per nucleotide) by following the appearance of free dye released from chromatin and DNA during thermal denaturation. The AMD dissociation profiles closely resembled the DNA or chromatin-DNA denaturation profiles; whereas the EB derivative dissociation profiles, indicated 3 major transitions for transcriptionally active chromatin with the main component corresponding to the single component which characterizes DNA. The DNA-like component was greatly reduced for mature erythrocyte chromatin but could be generated by removal of histone I and V. Removal of residual non acid-soluble proteins from dehistonized chromatin, urea treatment or dissociation and reconstitution of chromatin favoured conversion to the DNA-like component with loss of the other two. This study indicates that more than one type of binding exists generally in chromatin.

  19. Rescue of ligand binding of a mutant IGF-I receptor by complementation

    DEFF Research Database (Denmark)

    Chakravarty, Arjun Anders; Hinrichsen, Jane; Whittaker, Linda;

    2005-01-01

    The IGF-I receptor binds IGF-I with complex kinetics characterized by a curvilinear Scatchard plot, suggesting receptor heterogeneity and apparent negative cooperativity. To explore the molecular mechanisms underlying these properties, we have characterized the binding of a hybrid receptor formed...

  20. ncDNA and drift drive binding site accumulation

    Directory of Open Access Journals (Sweden)

    Ruths Troy

    2012-08-01

    Full Text Available Abstract Background The amount of transcription factor binding sites (TFBS in an organism’s genome positively correlates with the complexity of the regulatory network of the organism. However, the manner by which TFBS arise and accumulate in genomes and the effects of regulatory network complexity on the organism’s fitness are far from being known. The availability of TFBS data from many organisms provides an opportunity to explore these issues, particularly from an evolutionary perspective. Results We analyzed TFBS data from five model organisms – E. coli K12, S. cerevisiae, C. elegans, D. melanogaster, A. thaliana – and found a positive correlation between the amount of non-coding DNA (ncDNA in the organism’s genome and regulatory complexity. Based on this finding, we hypothesize that the amount of ncDNA, combined with the population size, can explain the patterns of regulatory complexity across organisms. To test this hypothesis, we devised a genome-based regulatory pathway model and subjected it to the forces of evolution through population genetic simulations. The results support our hypothesis, showing neutral evolutionary forces alone can explain TFBS patterns, and that selection on the regulatory network function does not alter this finding. Conclusions The cis-regulome is not a clean functional network crafted by adaptive forces alone, but instead a data source filled with the noise of non-adaptive forces. From a regulatory perspective, this evolutionary noise manifests as complexity on both the binding site and pathway level, which has significant implications on many directions in microbiology, genetics, and synthetic biology.

  1. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    Science.gov (United States)

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  2. Thermal denaturation of beta-galactosidase and of two site-specific mutants.

    Science.gov (United States)

    Edwards, R A; Jacobson, A L; Huber, R E

    1990-12-11

    The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.

  3. A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library.

    Science.gov (United States)

    Jain, Pankaj C; Varadarajan, Raghavan

    2014-03-15

    With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing.

  4. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    Institute of Scientific and Technical Information of China (English)

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

  5. L-(TH)glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Monaghan, D.T.; Yao, D.; Cotman, C.W.

    1985-08-12

    The anatomical distribution of L-(TH)glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-(TH)glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of CaS , Cl and Na ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-(TH)2-amino-5-phosphonopentanoate (D-(TH)AP5), (TH)kainate ((TH)KA) and (TH) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((TH)AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively. (Auth.). 29 refs.; 1 figure; 1 table.

  6. APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner.

    Science.gov (United States)

    Cun, Yanping; Dai, Nan; Li, Mengxia; Xiong, Chengjie; Zhang, Qinhong; Sui, Jiangdong; Qian, Chengyuan; Wang, Dong

    2014-02-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.

  7. A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

    Directory of Open Access Journals (Sweden)

    Emanuela Leonardi

    Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

  8. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

    OpenAIRE

    1999-01-01

    To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles expre...

  9. Novel Vinculin Binding Site of the IpaA Invasin of Shigella

    Energy Technology Data Exchange (ETDEWEB)

    Park, HaJeung; Valencia-Gallardo, Cesar; Sharff, Andrew; Van Nhieu, Guy Tran; Izard, Tina (Globel Phasing); (Scripps); (CF)

    2012-10-25

    Internalization of Shigella into host epithelial cells, where the bacteria replicates and spreads to neighboring cells, requires a type 3 secretion system (T3SS) effector coined IpaA. IpaA binds directly to and activates the cytoskeletal protein vinculin after injection in the host cell cytosol, and this was previously thought to be directed by two amphipathic {alpha}-helical vinculin-binding sites (VBS) found in the C-terminal tail domain of IpaA. Here, we report a third VBS, IpaA-VBS3, that is located N-terminal to the other two VBSs of IpaA and show that one IpaA molecule can bind up to three vinculin molecules. Biochemical in vitro Shigella invasion assays and the 1.6 {angstrom} crystal structure of the vinculin {center_dot} IpaA-VBS3 complex showed that IpaA-VBS3 is functionally redundant with the other two IpaA-VBSs in cell invasion and in activating the latent F-actin binding functions of vinculin. Multiple VBSs in IpaA are reminiscent of talin, which harbors 11 VBSs. However, most of the talin VBSs have low affinity and are buried in helix bundles, whereas all three of the VBSs of IpaA are high affinity, readily available, and in close proximity to each other in the IpaA structure. Although deletion of IpaA-VBS3 has no detectable effects on Shigella invasion of epithelial cells, deletion of all three VBSs impaired bacterial invasion to levels found in an ipaA null mutant strain. Thus, IpaA-directed mimicry of talin in activating vinculin occurs through three high affinity VBSs that are essential for Shigella pathogenesis.

  10. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-09-02

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

  11. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Fortin Sébastien

    2010-04-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  12. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Moreau Emmanuel

    2010-01-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  13. Prediction of nucleosome positioning based on transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

  14. Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

    Science.gov (United States)

    Winter, Jon J G; Anderson, Malcolm; Blades, Kevin; Brassington, Claire; Breeze, Alexander L; Chresta, Christine; Embrey, Kevin; Fairley, Gary; Faulder, Paul; Finlay, M Raymond V; Kettle, Jason G; Nowak, Thorsten; Overman, Ross; Patel, S Joe; Perkins, Paula; Spadola, Loredana; Tart, Jonathan; Tucker, Julie A; Wrigley, Gail

    2015-03-12

    Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

  15. 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

  16. Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

    Science.gov (United States)

    Herrero, S; González-Cabrera, J; Tabashnik, B E; Ferré, J

    2001-12-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.

  17. Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.

    Science.gov (United States)

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kopylov, A M

    2010-07-01

    In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

  18. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    Science.gov (United States)

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  19. Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant.

    Directory of Open Access Journals (Sweden)

    Khaled Barakat

    Full Text Available BACKGROUND: The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298-306 K. METHODOLOGY/PRINCIPAL FINDINGS: This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately. CONCLUSIONS: The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.

  20. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    Science.gov (United States)

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  1. Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

    Science.gov (United States)

    Suji, George; Khedkar, Santosh A; Singh, Sreelekha K; Kishore, Nand; Coutinho, Evans C; Bhor, Vikrant M; Sivakami, S

    2008-06-01

    The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

  2. Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum.

    Science.gov (United States)

    Matsuda, Keiko; Kondo, Tetsuro; Iijima, Takatoshi; Matsuda, Shinji; Watanabe, Masahiko; Yuzaki, Michisuke

    2009-02-01

    Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.

  3. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J;

    1993-01-01

    can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site.......(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus......Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching tRNA...

  4. Peripheral benzodiazepine binding sites on striated muscles of the rat: Properties and effect of denervation

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, W.E.; Ickstadt, A. (Mainz Univ. (Germany, F.R.). Pharmakologisches Inst.); Hopf, H.Ch. (Mainz Univ. (Germany, F.R.))

    1985-01-01

    In order to test the hypothesis that peripheral benzodiazepine binding sites mediate some direct effects of benzodiazepines on striated muscles, the properties of specific /sup 3/H-Ro 5-4864 binding to rat biceps and rat diaphragm homogenates were investigated. In both tissues a single population of sites was found with a Ksub(D) value of 3 nmol/l. The density of these sites in both muscles was higher than the density in rat brain, but was considerably lower than in rat kidney. Competition experiments indicate a substrate specificity of specific /sup 3/H-Ro 5-4864 binding similar to the properties already demonstrated for the specific binding of this ligand to peripheral benzodiazepine binding sites in many other tissues. The properties of these sites in the rat diaphragm are not changed after motoric denervation by phrenicectomy. It is concluded that peripheral benzodiazepine binding sites are not involved in direct effects of benzodiazepines on striated muscles.

  5. In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

    Directory of Open Access Journals (Sweden)

    Nima Najand

    Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

  6. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  7. Evolutionary computation for discovery of composite transcription factor binding sites

    Science.gov (United States)

    Fogel, Gary B.; Porto, V. William; Varga, Gabor; Dow, Ernst R.; Craven, Andrew M.; Powers, David M.; Harlow, Harry B.; Su, Eric W.; Onyia, Jude E.; Su, Chen

    2008-01-01

    Previous research demonstrated the use of evolutionary computation for the discovery of transcription factor binding sites (TFBS) in promoter regions upstream of coexpressed genes. However, it remained unclear whether or not composite TFBS elements, commonly found in higher organisms where two or more TFBSs form functional complexes, could also be identified by using this approach. Here, we present an important refinement of our previous algorithm and test the identification of composite elements using NFAT/AP-1 as an example. We demonstrate that by using appropriate existing parameters such as window size, novel-scoring methods such as central bonusing and methods of self-adaptation to automatically adjust the variation operators during the evolutionary search, TFBSs of different sizes and complexity can be identified as top solutions. Some of these solutions have known experimental relationships with NFAT/AP-1. We also indicate that even after properly tuning the model parameters, the choice of the appropriate window size has a significant effect on algorithm performance. We believe that this improved algorithm will greatly augment TFBS discovery. PMID:18927103

  8. Prediction of P53 mutants (multiple sites transcriptional activity based on structural (2D&3D properties.

    Directory of Open Access Journals (Sweden)

    R Geetha Ramani

    Full Text Available Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis.

  9. Multiplicity of carbohydrate-binding sites in -prism fold lectins: occurrence and possible evolutionary implications

    Indian Academy of Sciences (India)

    Alok Sharma; Divya Chandran; Desh D Singh; M Vijayan

    2007-09-01

    The -prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, -prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the -prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a -prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of -prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the -prism II fold, is related to the role of plant lectins in defence.

  10. Determination of energies and sites of binding of PFOA and PFOS to human serum albumin.

    Science.gov (United States)

    Salvalaglio, Matteo; Muscionico, Isabella; Cavallotti, Carlo

    2010-11-25

    Structure and energies of the binding sites of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to human serum albumin (HSA) were determined through molecular modeling. The calculations consisted of a compound approach based on docking, followed by molecular dynamics simulations and by the estimation of the free binding energies adopting WHAM-umbrella sampling and semiempirical methodologies. The binding sites so determined are common either to known HSA fatty acids sites or to other HSA sites known to bind to pharmaceutical compounds such as warfarin, thyroxine, indole, and benzodiazepin. Among the PFOA binding sites, five have interaction energies in excess of -6 kcal/mol, which become nine for PFOS. The calculated binding free energy of PFOA to the Trp 214 binding site is the highest among the PFOA complexes, -8.0 kcal/mol, in good agreement with literature experimental data. The PFOS binding site with the highest energy, -8.8 kcal/mol, is located near the Trp 214 binding site, thus partially affecting its activity. The maximum number of ligands that can be bound to HSA is 9 for PFOA and 11 for PFOS. The calculated data were adopted to predict the level of complexation of HSA as a function of the concentration of PFOA and PFOS found in human blood for different levels of exposition. The analysis of the factors contributing to the complex binding energy permitted to outline a set of guidelines for the rational design of alternative fluorinated surfactants with a lower bioaccumulation potential.

  11. ABS-Scan: In silico alanine scanning mutagenesis for binding site residues in protein-ligand complex.

    Science.gov (United States)

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most physiological processes in living systems are fundamentally regulated by protein-ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are calculated by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide a ranked list of residues to the user in order to obtain loss-of-function mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.

  12. Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5.

    Science.gov (United States)

    Hall, Spencer E; Mao, Allen; Nicolaidou, Vicky; Finelli, Mattea; Wise, Emma L; Nedjai, Belinda; Kanjanapangka, Julie; Harirchian, Paymann; Chen, Deborah; Selchau, Victor; Ribeiro, Sofia; Schyler, Sabine; Pease, James E; Horuk, Richard; Vaidehi, Nagarajan

    2009-06-01

    Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzohepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium (TAK-779), and a CCR2-specific antagonist N-(carbamoylmethyl)-3-trifluoromethyl benzamido-parachlorobenzyl 3-aminopyrrolidine (Teijin compound 1) in an ensemble of predicted structures of human CCR2 and CCR5. Based on our predictions of the protein-ligand interactions, we examined the activity of the antagonists for cells expressing thirteen mutants of CCR2 and five mutants of CCR5. The results show that residues Trp98(2.60) and Thr292(7.40) contribute significantly to the efficacy of both TAK-779 and Teijin compound 1, whereas His121(3.33) and Ile263(6.55) contribute significantly only to the antagonistic effect of Teijin compound 1 at CCR2. Mutation of residues Trp86(2.60) and Tyr108(3.32) adversely affected the efficacy of TAK-779 in antagonizing CCR5-mediated chemotaxis. Y49A(1.39) and E291A(7.39) mutants of CCR2 showed a complete loss of CCL2 binding and chemotaxis, despite robust cell surface expression, suggesting that these residues are critical in maintaining the correct receptor architecture. Modeling studies support the hypothesis that the residues Tyr49(1.39), Trp98(2.60), Tyr120(3.32), and Glu291(7.39) of CCR2 form a tight network of aromatic cluster and polar contacts between transmembrane helices 1, 2, 3, and 7.

  13. Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2

    Directory of Open Access Journals (Sweden)

    Shaun Martin

    2016-01-01

    Full Text Available The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9 is implicated in Parkinson’s disease (PD and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA and phosphatidylinositol (3,5 bisphosphate (PI(3,5P2, which modulate ATP13A2 activity under cellular stress conditions. Here, we analyzed stable human SHSY5Y cell lines overexpressing wild-type (WT or ATP13A2 mutants in which three N-terminal lipid binding sites (LBS1–3 were mutated. We explored the regulatory role of LBS1–3 in the cellular protection by ATP13A2 against mitochondrial stress induced by rotenone and found that the LBS2-3 mutants displayed an abrogated protective effect. Moreover, in contrast to WT, the LBS2 and LBS3 mutants responded poorly to pharmacological inhibition of, respectively, PI(3,5P2 and PA formation. We further demonstrate that PA and PI(3,5P2 are also required for the ATP13A2-mediated protection against the toxic metals Mn2+, Zn2+, and Fe3+, suggesting a general lipid-dependent activation mechanism of ATP13A2 in various PD-related stress conditions. Our results indicate that the ATP13A2-mediated protection requires binding of PI(3,5P2 to LBS2 and PA to LBS3. Thus, targeting the N-terminal lipid binding sites of ATP13A2 might offer a therapeutic approach to reduce cellular toxicity of various PD insults including mitochondrial stress.

  14. Characterization of the hydrophobic substrate-binding site of the bacterial beta class glutathione transferase from Proteus mirabilis.

    Science.gov (United States)

    Federici, Luca; Masulli, Michele; Di Ilio, Carmine; Allocati, Nerino

    2010-09-01

    Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes.

  15. Prediction of calcium-binding sites by combining loop-modeling with machine learning

    Directory of Open Access Journals (Sweden)

    Altman Russ B

    2009-12-01

    Full Text Available Abstract Background Protein ligand-binding sites in the apo state exhibit structural flexibility. This flexibility often frustrates methods for structure-based recognition of these sites because it leads to the absence of electron density for these critical regions, particularly when they are in surface loops. Methods for recognizing functional sites in these missing loops would be useful for recovering additional functional information. Results We report a hybrid approach for recognizing calcium-binding sites in disordered regions. Our approach combines loop modeling with a machine learning method (FEATURE for structure-based site recognition. For validation, we compared the performance of our method on known calcium-binding sites for which there are both holo and apo structures. When loops in the apo structures are rebuilt using modeling methods, FEATURE identifies 14 out of 20 crystallographically proven calcium-binding sites. It only recognizes 7 out of 20 calcium-binding sites in the initial apo crystal structures. We applied our method to unstructured loops in proteins from SCOP families known to bind calcium in order to discover potential cryptic calcium binding sites. We built 2745 missing loops and evaluated them for potential calcium binding. We made 102 predictions of calcium-binding sites. Ten predictions are consistent with independent experimental verifications. We found indirect experimental evidence for 14 other predictions. The remaining 78 predictions are novel predictions, some with intriguing potential biological significance. In particular, we see an enrichment of beta-sheet folds with predicted calcium binding sites in the connecting loops on the surface that may be important for calcium-mediated function switches. Conclusion Protein crystal structures are a potentially rich source of functional information. When loops are missing in these structures, we may be losing important information about binding sites and active

  16. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    Science.gov (United States)

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G

    1998-06-12

    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  17. An internal ribosome entry site (IRES mutant library for tuning expression level of multiple genes in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Esther Y C Koh

    Full Text Available A set of mutated Encephalomyocarditis virus (EMCV internal ribosome entry site (IRES elements with varying strengths is generated by mutating the translation initiation codons of 10(th, 11(th, and 12(th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

  18. T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    Directory of Open Access Journals (Sweden)

    Zhuang Fanglei

    2011-07-01

    Full Text Available Abstract Background T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr, is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA. Results With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction. Conclusions Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.

  19. Structural Perspectives on the Evolutionary Expansion of Unique Protein-Protein Binding Sites.

    Science.gov (United States)

    Goncearenco, Alexander; Shaytan, Alexey K; Shoemaker, Benjamin A; Panchenko, Anna R

    2015-09-15

    Structures of protein complexes provide atomistic insights into protein interactions. Human proteins represent a quarter of all structures in the Protein Data Bank; however, available protein complexes cover less than 10% of the human proteome. Although it is theoretically possible to infer interactions in human proteins based on structures of homologous protein complexes, it is still unclear to what extent protein interactions and binding sites are conserved, and whether protein complexes from remotely related species can be used to infer interactions and binding sites. We considered biological units of protein complexes and clustered protein-protein binding sites into similarity groups based on their structure and sequence, which allowed us to identify unique binding sites. We showed that the growth rate of the number of unique binding sites in the Protein Data Bank was much slower than the growth rate of the number of structural complexes. Next, we investigated the evolutionary roots of unique binding sites and identified the major phyletic branches with the largest expansion in the number of novel binding sites. We found that many binding sites could be traced to the universal common ancestor of all cellular organisms, whereas relatively few binding sites emerged at the major evolutionary branching points. We analyzed the physicochemical properties of unique binding sites and found that the most ancient sites were the largest in size, involved many salt bridges, and were the most compact and least planar. In contrast, binding sites that appeared more recently in the evolution of eukaryotes were characterized by a larger fraction of polar and aromatic residues, and were less compact and more planar, possibly due to their more transient nature and roles in signaling processes.

  20. Oestradiol and testosterone binding sites in mice tibiae and their relationship with bone growth.

    Science.gov (United States)

    Lopez, A; Ventanas, J; Burgos, J

    1986-11-01

    High affinity oestradiol and testosterone binding sites were found in tibiae cytosol from entire male and female of different ages. Scatchard assay allowed to estimate a Kd of 2.7 X 10(-9) M for oestradiol binding sites indicating that the 3H-oestradiol binding was of high affinity. Oestradiol and testosterone binding sites abundance in mice tibiae are subject to change with age. It is not easy to establish a direct correlation between these changes and the values reported here on bone growth in weight and length, however seems possible to point a negative relationship between bone lengthening and oestradiol binding site levels in female, as well a positive relationship with testosterone in both sexes. The presence of oestradiol and testosterone binding sites in epiphyses and not in the diaphyses reinforces the hypothesis that both are playing some role in bone growth.

  1. Alignment-free ultra-high-throughput comparison of druggable protein-ligand binding sites.

    Science.gov (United States)

    Weill, Nathanaël; Rognan, Didier

    2010-01-01

    Inferring the biological function of a protein from its three-dimensional structure as well as explaining why a drug may bind to various targets is of crucial importance to modern drug discovery. Here we present a generic 4833-integer vector describing druggable protein-ligand binding sites that can be applied to any protein and any binding cavity. The fingerprint registers counts of pharmacophoric triplets from the Calpha atomic coordinates of binding-site-lining residues. Starting from a customized data set of diverse protein-ligand binding site pairs, the most appropriate metric and a similarity threshold could be defined for similar binding sites. The method (FuzCav) has been used in various scenarios: (i) screening a collection of 6000 binding sites for similarity to different queries; (ii) classifying protein families (serine endopeptidases, protein kinases) by binding site diversity; (iii) discriminating adenine-binding cavities from decoys. The fingerprint generation and comparison supports ultra-high throughput (ca. 1000 measures/s), does not require prior alignment of protein binding sites, and is able to detect local similarity among subpockets. It is thus particularly well suited to the functional annotation of novel genomic structures with low sequence identity to known X-ray templates.

  2. Identification of a second substrate-binding site in solute-sodium symporters.

    Science.gov (United States)

    Li, Zheng; Lee, Ashley S E; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P; Abramson, Jeff; Quick, Matthias; Shi, Lei

    2015-01-02

    The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.

  3. Characterization of metal binding in the active sites of acireductone dioxygenase isoforms from Klebsiella ATCC 8724.

    Science.gov (United States)

    Chai, Sergio C; Ju, Tingting; Dang, Marina; Goldsmith, Rachel Beaulieu; Maroney, Michael J; Pochapsky, Thomas C

    2008-02-26

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but

  4. Identification of clustered YY1 binding sites in Imprinting Control Regions

    Energy Technology Data Exchange (ETDEWEB)

    Kim, J D; Hinz, A; Bergmann, A; Huang, J; Ovcharenko, I; Stubbs, L; Kim, J

    2006-04-19

    Mammalian genomic imprinting is regulated by Imprinting Control Regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In the current study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele-specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.

  5. Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless.

    Directory of Open Access Journals (Sweden)

    Patricia Kurth

    Full Text Available The Notch signalling pathway mediates cell-cell communication in a wide variety of organisms. The major components, as well as the basic mechanisms of Notch signal transduction, are remarkably well conserved amongst vertebrates and invertebrates. Notch signalling results in transcriptional activation of Notch target genes, which is mediated by an activator complex composed of the DNA binding protein CSL, the intracellular domain of the Notch receptor, and the transcriptional coactivator Mastermind. In the absence of active signalling, CSL represses transcription from Notch target genes by the recruitment of corepressors. The Notch activator complex is extremely well conserved and has been studied in great detail. However, Notch repressor complexes are far less understood. In Drosophila melanogaster, the CSL protein is termed Suppressor of Hairless [Su(H]. Su(H functions as a transcriptional repressor by binding Hairless, the major antagonist of Notch signalling in Drosophila, which in turn recruits two general corepressors--Groucho and C-terminal binding protein CtBP. Recently, we determined that the C-terminal domain (CTD of Su(H binds Hairless and identified a single site in Hairless, which is essential for contacting Su(H. Here we present additional biochemical and in vivo studies aimed at mapping the residues in Su(H that contact Hairless. Focusing on surface exposed residues in the CTD, we identified two sites that affect Hairless binding in biochemical assays. Mutation of these sites neither affects binding to DNA nor to Notch. Subsequently, these Su(H mutants were found to function normally in cellular and in vivo assays using transgenic flies. However, these experiments rely on Su(H overexpression, which does not allow for detection of quantitative or subtle differences in activity. We discuss the implications of our results.

  6. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  7. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  8. Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

    Science.gov (United States)

    Nim, Shweta; Lobato, Lucia Gonzalez; Moreno, Alexis; Chaptal, Vincent; Rawal, Manpreet Kaur; Falson, Pierre; Prasad, Rajendra

    2016-11-01

    The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca(2+) ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

  9. Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.; Goberna, R.; Guerrero, J.M. (Univ. of Seville School of Medicine, Sevilla (Spain))

    1991-01-01

    The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using ({sup 125}I)melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37{degree}C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of ({sup 125}I)melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of ({sup 125}I)melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the ({sup 125}I)melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.

  10. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska;

    2016-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently...

  11. Genetic probes of structure/function relationships in the Q{sub B} binding site of the photosynthetic reaction center

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, D.K.; Tiede, D.M.; Nance, S.L.; Chang, Chong-Hwan; Schiffer, M.

    1991-06-25

    In photosynthetic reaction centers, a quinone molecule, Q{sub B}, is the terminal acceptor in light-induced electron transfer. The crystal structure of the reaction center implicates the protonatable amiho acid residues L212Glu and L213Asp in the binding of Q{sub B} to the reaction center and in proton transfer to the anionic forms of Q{sub B} generated by electron transfer from Q{sub A}. Here we report the construction of the double mutant L212Ala-L213Ala by site-specific mutagenesis, and the isolation and preliminary biophysical characterization of revertant and suppressor strains that have regained the ability to grow under photosynthetic conditions. Our results show that neither L212Glu nor L213Asp is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus and that second-site mutations, located within the QB binding pocket or at a more distant site, can compensate for mutations at L212 and L213. Acquisition of a single negatively charged residue (at position L213, or on the other side of the binding pocket at position L225) or loss of a positively charged residue (at position M231) is sufficient to restore activity to the complex.

  12. Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

    OpenAIRE

    Borgeson, Claudia D.; Samson, Marie-Laure

    2005-01-01

    The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified prev...

  13. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    Energy Technology Data Exchange (ETDEWEB)

    Roman, F.; Pascaud, X.; Vauche, D.; Junien, J.

    1988-01-01

    The presence of a binding site to (+)-(/sup 3/H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10/sup -4/M were unable to displace (+)-(/sup 3/H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.

  14. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    Science.gov (United States)

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

  15. Does transcription play a role in creating a condensin binding site?

    Science.gov (United States)

    Bernard, Pascal; Vanoosthuyse, Vincent

    2015-01-01

    The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive. Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

  16. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  17. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.

    Science.gov (United States)

    Seidel, Susanne A I; Wienken, Christoph J; Geissler, Sandra; Jerabek-Willemsen, Moran; Duhr, Stefan; Reiter, Alwin; Trauner, Dirk; Braun, Dieter; Baaske, Philipp

    2012-10-15

    Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca(2+) ions to synaptotagmin was quantified.

  18. Identification of multiple SNT-binding sites on NPM-ALK oncoprotein and their involvement in cell transformation.

    Science.gov (United States)

    Chikamori, M; Fujimoto, J; Tokai-Nishizumi, N; Yamamoto, T

    2007-05-01

    The t(2;5) chromosomal translocation occurs in anaplastic large-cell lymphoma arising from activated T lymphocytes. This genomic rearrangement generates the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) oncoprotein that is a chimeric protein consisting of parts of the nuclear protein NPM and ALK receptor protein-tyrosine kinase. We used yeast two-hybrid screening to identify an adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT)-2 as a new partner that interacted with the cytoplasmic domain of ALK. Immunoprecipitation assay revealed that SNT-1 and SNT-2 interacted with NPM-ALK and kinase-negative NPM-ALK mutant. Y156, Y567 and a 19-amino-acid sequence (aa 631-649) of NPM-ALK were essential for this interaction. The interaction through Y156 and Y567 was dependent on phosphorylation of these tyrosines, whereas the interaction through the 19-amino-acid sequence was independent of phosphorylation. NPM-ALK mutant protein mutated at these three binding sites showed significantly reduced transforming activity. This transformation-defective NPM-ALK mutant still interacted with signal transducing proteins such as phospholipase C-gamma and phosphatidylinositol 3-kinase, which were previously reported to be relevant to NPM-ALK-dependent tumorigenesis. These observations indicate that the three SNT-binding sites of NPM-ALK are important for its transforming activity. This raises a possibility that SNT family proteins play significant roles in cellular transformation triggered by NPM-ALK, which though remains to be verified.

  19. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tokutomi, Y; Takenaka, H; Hamada, M; Kuby, S A; Uesugi, S

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

  20. Spatial distribution of predicted transcription factor binding sites in Drosophila ChIP peaks.

    Science.gov (United States)

    Pettie, Kade P; Dresch, Jacqueline M; Drewell, Robert A

    2016-08-01

    In the development of the Drosophila embryo, gene expression is directed by the sequence-specific interactions of a large network of protein transcription factors (TFs) and DNA cis-regulatory binding sites. Once the identity of the typically 8-10bp binding sites for any given TF has been determined by one of several experimental procedures, the sequences can be represented in a position weight matrix (PWM) and used to predict the location of additional TF binding sites elsewhere in the genome. Often, alignments of large (>200bp) genomic fragments that have been experimentally determined to bind the TF of interest in Chromatin Immunoprecipitation (ChIP) studies are trimmed under the assumption that the majority of the binding sites are located near the center of all the aligned fragments. In this study, ChIP/chip datasets are analyzed using the corresponding PWMs for the well-studied TFs; CAUDAL, HUNCHBACK, KNIRPS and KRUPPEL, to determine the distribution of predicted binding sites. All four TFs are critical regulators of gene expression along the anterio-posterior axis in early Drosophila development. For all four TFs, the ChIP peaks contain multiple binding sites that are broadly distributed across the genomic region represented by the peak, regardless of the prediction stringency criteria used. This result suggests that ChIP peak trimming may exclude functional binding sites from subsequent analyses.

  1. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

    Full Text Available Bisphenol A (BPA has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.

  2. Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2.

    Science.gov (United States)

    Leitinger, Birgit

    2003-05-09

    The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.

  3. Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase.

    Science.gov (United States)

    Zhang, Xiaohua; Caner, Sami; Kwan, Emily; Li, Chunmin; Brayer, Gary D; Withers, Stephen G

    2016-11-01

    Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.

  4. Characterization of the binding sites for dicarboxylic acids on bovine serum albumin.

    Science.gov (United States)

    Tonsgard, J H; Meredith, S C

    1991-06-15

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic.

  5. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    Science.gov (United States)

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  6. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    Directory of Open Access Journals (Sweden)

    Ionas Erb

    Full Text Available The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1 occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  7. [Type-I and -II estradiol binding sites in the endometrium during blastocyst implantation].

    Science.gov (United States)

    Bernal, A; Calzada, L; Hicks, J J; Velázquez, A

    1989-04-01

    The properties of type I and occupied and unoccupied type II cytosolic estrogen binding sites in the rat endometrium were analyzed on day five of pregnancy; the samples studied correspond to blastocyst receptive endometrium (implantation sites), nonreceptive endometrium and ovariectomized uterine horn endometrium, from the same pregnancy rats. The occupied binding site type II was analyzed by exchange assays. Dissociation constant obtained from experiments carried out at 4 or 25 degrees C are similar for each one of the binding site at the three different endometrium samples; the binding capacity (femtomoles/mg protein) from the sites type I and type II and the ratio between occupied (by endogenous estradiol) and unoccupied site type II, seems to be characteristic for each one of the three analyzed endometrium.

  8. Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Lin, A P; McCammon, M T; McAlister-Henn, L

    2001-11-27

    Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic

  9. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  10. Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

    Science.gov (United States)

    Hübner, S; Eam, J E; Hübner, A; Jans, D A

    2006-01-15

    Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

  11. Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species.

    Directory of Open Access Journals (Sweden)

    Robert K Bradley

    2010-03-01

    Full Text Available Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances.

  12. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    NARCIS (Netherlands)

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

  13. DO-RIP-seq to quantify RNA binding sites transcriptome-wide.

    Science.gov (United States)

    Nicholson, Cindo O; Friedersdorf, Matthew B; Bisogno, Laura S; Keene, Jack D

    2016-11-10

    Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.

  14. Dynamic fluorescence spectroscopy on single tryptophan mutants of EIImtl in detergent micelles : Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay

    NARCIS (Netherlands)

    Swaving Dijkstra, Dolf; Broos, J.; Visser, Antonie J.W.G.; van Hoek, A.; Robillard, George

    1997-01-01

    The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C d

  15. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Crankshaw, D.; Gaspar, V.; Pliska, V. (McMaster Univ., Hamilton, Ontario, (Canada))

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  16. Impact of Binding Site Comparisons on Medicinal Chemistry and Rational Molecular Design.

    Science.gov (United States)

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods.

  17. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin.

    Science.gov (United States)

    Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity.

  18. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    Science.gov (United States)

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

  19. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  20. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models.

    Science.gov (United States)

    Mehta, Pankaj; Schwab, David J; Sengupta, Anirvan M

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

  1. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    OpenAIRE

    Mashiur Rahman; Farzana Prianka; Mohammad Shohel; Md. Abdul Mazid

    2014-01-01

    Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA) was studied by equilibrium dialysis method (ED) at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 a...

  2. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

    Directory of Open Access Journals (Sweden)

    Oscar Harari

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  3. Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

    Directory of Open Access Journals (Sweden)

    Joel Raborn

    Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

  4. Enhanced human receptor binding by H5 haemagglutinins

    OpenAIRE

    Xiong, Xiaoli; Xiao, Haixia; Martin, Stephen R.; Coombs, Peter J.; Liu, Junfeng; Collins, Patrick J.; Vachieri, Sebastien G.; Walker, Philip A.; Lin, Yi Pu; McCauley, John W.; Gamblin, Steven J.; John J Skehel

    2014-01-01

    Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between As...

  5. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites...

  6. Partial enterectomy decreases somatostatin-binding sites in residual intestine of rabbits.

    Science.gov (United States)

    Colas, B; Bodegas, G; Sanz, M; Prieto, J C; Arilla, E

    1988-05-01

    1. Three weeks after partial enterectomy in the rabbit there was an increased somatostatin concentration and a decreased number of somatostatin-binding sites (without changes in the corresponding affinity values) in the cytosol of the residual intestinal tissue, except in the terminal ileum and the colon. 2. Five weeks after surgery both the somatostatin concentration and the number of somatostatin-binding sites returned towards control values. 3. These results suggest that an increase in bowel somatostatin content could lead to down-regulation of somatostatin-binding sites in the intestinal mucosa.

  7. Brominated lipids identify lipid binding sites on the surface of the reaction center from Rhodobacter sphaeroides.

    Science.gov (United States)

    Roszak, Aleksander W; Gardiner, Alastair T; Isaacs, Neil W; Cogdell, Richard J

    2007-03-20

    This study describes the use of brominated phospholipids to distinguish between lipid and detergent binding sites on the surface of a typical alpha-helical membrane protein. Reaction centers isolated from Rhodobacter sphaeroides were cocrystallized with added brominated phospholipids. X-ray structural analysis of these crystals has revealed the presence of two lipid binding sites from the characteristic strong X-ray scattering from the bromine atoms. These results demonstrate the usefulness of this approach to mapping lipid binding sites at the surface of membrane proteins.

  8. Identification of ligands that target the HCV-E2 binding site on CD81

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M.; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  9. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    Science.gov (United States)

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  10. Identification of ligands that target the HCV-E2 binding site on CD81.

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  11. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site.

    Science.gov (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-03-28

    Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

  12. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Science.gov (United States)

    den-Haan, Helena; Pérez-Sánchez, Horacio; Del Prete, Rosita; Liguori, Ludovica; Cimmaruta, Chiara; Lukas, Jan; Andreotti, Giuseppina

    2016-01-01

    Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. PMID:27788225

  13. An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

    Institute of Scientific and Technical Information of China (English)

    Bao-zhong ZHANG; Xin ZHANG; Xiao-ping AN; Duo-liang RAN; Yu-sen ZHOU; Jun LU; Yi-gang TONG

    2009-01-01

    Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.

  14. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

    Directory of Open Access Journals (Sweden)

    Thomas L Prince

    Full Text Available The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

  15. Europium ion as a probe for binding sites to carrageenans

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil); Zaniquelli, Maria Elisabete D. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil)], E-mail: medzaniquelli@ffclrp.usp.br; Wong, Kenneth [Laboratorio de Fisico-Quimica, Centro de Pesquisas de Paulinia, Rhodia Brasil, Paulinia, Sao Paulo (Brazil)

    2007-12-15

    Carrageenans, sulfated polysaccharides extracted from red algae, present a coil-helix transition and helix aggregation dependence on the type and concentration of counterions. In this study, we focus attention on a mixed valence counterion system: Eu{sup 3+}/Na{sup +} or K{sup +} with different gel-forming carrageenans: kappa, iota, and kappa-2. Results of stationary and time-dependent luminescence showed to be a suitable tool to probe ion binding to both the negatively charged sulfate group and the hydroxyl groups present in the biopolymer. For lower europium ion concentrations, a single longer decay emission lifetime was detected, which was attributed to the binding of europium ion to the carrageenan sulfate groups. An additional decay ascribed to europium binding to hydroxyl groups was observed above a threshold concentration, and this decay was dependent on the carrageenan charge density. Symmetry of the europium ion microenvironment was estimated by the ratio between the intensities of its emission bands, which has been shown to depend on the concentration of europium ions and on the specificity of the monovalent counterion bound to the carrageenan.

  16. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    Science.gov (United States)

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  17. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...... around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  18. Predicting DNA-binding sites of proteins based on sequential and 3D structural information.

    Science.gov (United States)

    Li, Bi-Qing; Feng, Kai-Yan; Ding, Juan; Cai, Yu-Dong

    2014-06-01

    Protein-DNA interactions play important roles in many biological processes. To understand the molecular mechanisms of protein-DNA interaction, it is necessary to identify the DNA-binding sites in DNA-binding proteins. In the last decade, computational approaches have been developed to predict protein-DNA-binding sites based solely on protein sequences. In this study, we developed a novel predictor based on support vector machine algorithm coupled with the maximum relevance minimum redundancy method followed by incremental feature selection. We incorporated not only features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, solvent accessibility, but also five three-dimensional (3D) structural features calculated from PDB data to predict the protein-DNA interaction sites. Feature analysis showed that 3D structural features indeed contributed to the prediction of DNA-binding site and it was demonstrated that the prediction performance was better with 3D structural features than without them. It was also shown via analysis of features from each site that the features of DNA-binding site itself contribute the most to the prediction. Our prediction method may become a useful tool for identifying the DNA-binding sites and the feature analysis described in this paper may provide useful insights for in-depth investigations into the mechanisms of protein-DNA interaction.

  19. Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

    2008-01-01

    with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit...... that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley alpha-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends...

  20. Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

    Science.gov (United States)

    Hanson, D K; Tiede, D M; Nance, S L; Chang, C H; Schiffer, M

    1993-01-01

    In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated proton transport. PMID:8105468

  1. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

  2. Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites

    DEFF Research Database (Denmark)

    Plenge, Per; Gether, Ulrik; Rasmussen, Søren G

    2007-01-01

    SERT and the three mutants. Further, R-citalopram previously thought of as an inactive enantiomer strongly attenuated dissociation of the wild-type [(3)H]-imipramine:hSERT complex, whereas S-citalopram had almost no effect on this complex. These results suggest that 1: The allosteric site on hSERT is distinct from...... the site to which S-citalopram binds with high affinity. 2: The allosteric effects of R-citalopram on the dissociation of [(3)H]-imipramine from hSERT indicate that R-citalopram introduces a conformational change in hSERT....

  3. Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

    2015-04-01

    Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.

  4. Characterization of the β-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

    NARCIS (Netherlands)

    Alkema, Wynand B.L.; Hensgens, Charles M.H.; Kroezinga, Els H.; de Vries, Erik; Floris, René; Laan, Jan-Metske van der; Dijkstra, Bauke W.; Janssen, Dick B.

    2000-01-01

    The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme–substrate complex was determined after soaking crystals of an inactive βN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in wh

  5. Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

    NARCIS (Netherlands)

    Alkema, WBL; Hensgens, CMH; Kroezinga, EH; de Vries, E; Floris, R; van der Laan, JM; Dijkstra, BW; Janssen, DB

    2000-01-01

    The binding of penicillin to penicillin acylase was studied by X-ray crystallography, The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive beta N241A penicillin acylase mutant with penicillin G, Binding of the substrate induces a conformational change, i

  6. Three-dimensional binding sites volume assessment during cardiac pacing lead extraction

    Directory of Open Access Journals (Sweden)

    Bich Lien Nguyen

    2015-07-01

    Conclusions: Real-time 3D binding sites assessment is feasible and improves transvenous lead extraction outcomes. Its role as a complementary information requires extensive validation, and might be beneficial for a tailored strategy.

  7. Femtosecond fluorescence spectra of tryptophan in human gamma-crystallin mutants: site-dependent ultrafast quenching.

    Science.gov (United States)

    Xu, Jianhua; Chen, Jiejin; Toptygin, Dmitri; Tcherkasskaya, Olga; Callis, Patrik; King, Jonathan; Brand, Ludwig; Knutson, Jay R

    2009-11-25

    The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

  8. Probing and mapping the binding sites on streptavidin imprinted polymer surface

    Energy Technology Data Exchange (ETDEWEB)

    Duman, Memed, E-mail: memi@hacettepe.edu.tr

    2014-10-01

    Molecular imprinting is an effective technique for preparing recognition sites which act as synthetic receptors on polymeric surfaces. Herein, we synthesized MIP surfaces with specific binding sites for streptavidin and characterized them at nanoscale by using two different atomic force microscopy (AFM) techniques. While the single molecule force spectroscopy (SMFS) reveals the unbinding kinetics between streptavidin molecule and binding sites, simultaneous topography and recognition imaging (TREC) was employed, for the first time, to directly map the binding sites on streptavidin imprinted polymers. Streptavidin modified AFM cantilever showed specific unbinding events with an unbinding force around 300 pN and the binding probability was calculated as 35.2% at a given loading rate. In order to prove the specificity of the interaction, free streptavidin molecules were added to AFM liquid cell and the binding probability was significantly decreased to 7.6%. Moreover, the recognition maps show that the smallest recognition site with a diameter of around ∼ 21 nm which corresponds to a single streptavidin molecule binding site. We believe that the potential of combining SMFS and TREC opens new possibilities for the characterization of MIP surfaces with single molecule resolution under physiological conditions. - Graphical abstract: Simultaneous Topography and RECognition (TREC) imaging is a novel characterization technique to reveal binding sites on molecularly imprinted polymer surfaces with single molecule resolution under physiological conditions. - Highlights: • Highly specific streptavidin printed polymer surfaces were synthesized. • Unbinding kinetic rate of single streptavidin molecule was studied by SMFS. • The distribution of binding pockets was revealed for the first time by TREC imaging. • TREC showed that the binding pockets formed nano-domains on MIP surface. • SMFS and TREC are powerful AFM techniques for characterization of MIP surfaces.

  9. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.;

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...... of 25 mu M, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 mu M and 2 mu M, respectively, The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics....

  10. Radiolabelling of phoneutria nigriventer spider toxin (Tx1): a tool to study its binding site

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Diniz, Carlos Roberto; Nascimento, Marta Cordeiro [FUNED, Belo Horizonte, MG (Brazil); Lima, Maria Elena de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    1996-07-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ({sup 125} I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na{sup 125} I by the lactoperoxidase method. {sup 125} I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10{sup -10} M) and low capacity (1.2 pmol/mg protein). The specific binding increased in parallel with the protein concentration. In competition experiments the ligands of ionic channels used (sodium, potassium and calcium) did not affect the binding of {sup 125} I-Tx1 to MPLM neither did the cholinergic ligands (hemicholinium-3, hexamethonium, d-tubocurarine and atropine). Another neurotoxin (Tx2-6, one of the isoforms of Tx2 pool) decreased toxin with MPLM and showed that toxin has a specific and saturable binding site in guinea pig ileum and this binding site appears to be related to the Tx2 site. (author)

  11. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  12. Identifying ligand binding sites and poses using GPU-accelerated Hamiltonian replica exchange molecular dynamics.

    Science.gov (United States)

    Wang, Kai; Chodera, John D; Yang, Yanzhi; Shirts, Michael R

    2013-12-01

    We present a method to identify small molecule ligand binding sites and poses within a given protein crystal structure using GPU-accelerated Hamiltonian replica exchange molecular dynamics simulations. The Hamiltonians used vary from the physical end state of protein interacting with the ligand to an unphysical end state where the ligand does not interact with the protein. As replicas explore the space of Hamiltonians interpolating between these states, the ligand can rapidly escape local minima and explore potential binding sites. Geometric restraints keep the ligands from leaving the vicinity of the protein and an alchemical pathway designed to increase phase space overlap between intermediates ensures good mixing. Because of the rigorous statistical mechanical nature of the Hamiltonian exchange framework, we can also extract binding free energy estimates for all putative binding sites. We present results of this methodology applied to the T4 lysozyme L99A model system for three known ligands and one non-binder as a control, using an implicit solvent. We find that our methodology identifies known crystallographic binding sites consistently and accurately for the small number of ligands considered here and gives free energies consistent with experiment. We are also able to analyze the contribution of individual binding sites to the overall binding affinity. Our methodology points to near term potential applications in early-stage structure-guided drug discovery.

  13. Effect of positional dependence and alignment strategy on modeling transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Quader Saad

    2012-07-01

    Full Text Available Abstract Background Many consensus-based and Position Weight Matrix-based methods for recognizing transcription factor binding sites (TFBS are not well suited to the variability in the lengths of binding sites. Besides, many methods discard known binding sites while building the model. Moreover, the impact of Information Content (IC and the positional dependence of nucleotides within an aligned set of TFBS has not been well researched for modeling variable-length binding sites. In this paper, we propose ML-Consensus (Mixed-Length Consensus: a consensus model for variable-length TFBS which does not exclude any reported binding sites. Methods We consider Pairwise Score (PS as a measure of positional dependence of nucleotides within an alignment of TFBS. We investigate how the prediction accuracy of ML-Consensus is affected by the incorporation of IC and PS with a particular binding site alignment strategy. We perform cross-validations for datasets of six species from the TRANSFAC public database, and analyze the results using ROC curves and the Wilcoxon matched-pair signed-ranks test. Results We observe that the incorporation of IC and PS in ML-Consensus results in statistically significant improvement in the prediction accuracy of the model. Moreover, the existence of a core region among the known binding sites (of any length is witnessed by the pairwise coexistence of nucleotides within the core length. Conclusions These observations suggest the possibility of an efficient multiple sequence alignment algorithm for aligning TFBS, accommodating known binding sites of any length, for optimal (or near-optimal TFBS prediction. However, designing such an algorithm is a matter of further investigation.

  14. Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Israel, A.; Correa, F.M.A.; Niwa, M.; Saavedra, J.M. (National Inst. of Mental Health, Bethesda, MD (USA))

    1984-11-26

    Rat brain and pituitary angiotensin II (AII) binding sites were quantitated by incubation of tissue sections with /sup 125/I-(Sar/sup 1/) AII, Ultrofilm radioautography, computerized densitometry, and comparison with /sup 125/I-standards at appropriate film exposure times. The highest number of AII binding sites was found in anterior pituitary and the circumventricular organs, organon subfornicalis and organon vasculosum laminae terminalis.

  15. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1984-01-01

    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

  16. A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.

    Science.gov (United States)

    Yu, J; Wang, C; Palmieri, S J; Haarer, B K; Field, J

    1999-07-09

    In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.

  17. In Silico Docking, Molecular Dynamics and Binding Energy Insights into the Bolinaquinone-Clathrin Terminal Domain Binding Site

    Directory of Open Access Journals (Sweden)

    Mohammed K. Abdel-Hamid

    2014-05-01

    Full Text Available Clathrin-mediated endocytosis (CME is a process that regulates selective internalization of important cellular cargo using clathrin-coated vesicles. Perturbation of this process has been linked to many diseases including cancer and neurodegenerative conditions. Chemical proteomics identified the marine metabolite, 2-hydroxy-5-methoxy-3-(((1S,4aS,8aS-1,4a,5-trimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-2-ylmethylcyclohexa- 2,5-diene-1,4-dione (bolinaquinone as a clathrin inhibitor. While being an attractive medicinal chemistry target, the lack of data about bolinaquinone’s mode of binding to the clathrin enzyme represents a major limitation for its structural optimization. We have used a molecular modeling approach to rationalize the observed activity of bolinaquinone and to predict its mode of binding with the clathrin terminal domain (CTD. The applied protocol started by global rigid-protein docking followed by flexible docking, molecular dynamics and linear interaction energy calculations. The results revealed the potential of bolinaquinone to interact with various pockets within the CTD, including the clathrin-box binding site. The results also highlight the importance of electrostatic contacts over van der Waals interactions for proper binding between bolinaquinone and its possible binding sites. This study provides a novel model that has the potential to allow rapid elaboration of bolinaquinone analogues as a new class of clathrin inhibitors.

  18. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  19. Cycloxaprid insecticide: nicotinic acetylcholine receptor binding site and metabolism.

    Science.gov (United States)

    Shao, Xusheng; Swenson, Tami L; Casida, John E

    2013-08-21

    Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

  20. Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site, Is Involved in Cation Selectivity, and Affects Cation-binding Site Cooperativity

    Science.gov (United States)

    Shea, Michael E.; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-01-01

    The Na+-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC). PMID:24030824

  1. MBSTAR: multiple instance learning for predicting specific functional binding sites in microRNA targets

    Science.gov (United States)

    Bandyopadhyay, Sanghamitra; Ghosh, Dip; Mitra, Ramkrishna; Zhao, Zhongming

    2015-01-01

    MicroRNA (miRNA) regulates gene expression by binding to specific sites in the 3'untranslated regions of its target genes. Machine learning based miRNA target prediction algorithms first extract a set of features from potential binding sites (PBSs) in the mRNA and then train a classifier to distinguish targets from non-targets. However, they do not consider whether the PBSs are functional or not, and consequently result in high false positive rates. This substantially affects the follow up functional validation by experiments. We present a novel machine learning based approach, MBSTAR (Multiple instance learning of Binding Sites of miRNA TARgets), for accurate prediction of true or functional miRNA binding sites. Multiple instance learning framework is adopted to handle the lack of information about the actual binding sites in the target mRNAs. Biologically validated 9531 interacting and 973 non-interacting miRNA-mRNA pairs are identified from Tarbase 6.0 and confirmed with PAR-CLIP dataset. It is found that MBSTAR achieves the highest number of binding sites overlapping with PAR-CLIP with maximum F-Score of 0.337. Compared to the other methods, MBSTAR also predicts target mRNAs with highest accuracy. The tool and genome wide predictions are available at http://www.isical.ac.in/~bioinfo_miu/MBStar30.htm.

  2. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer.

    Science.gov (United States)

    Kim, Jae-Young; Stewart, Paul A; Borne, Adam L; Fang, Bin; Welsh, Eric A; Chen, Yian Ann; Eschrich, Steven A; Koomen, John M; Haura, Eric B

    2016-06-01

    One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  3. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    2016-04-01

    Full Text Available One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  4. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  5. In vitro and in vivo characterisation of [3H]ANSTO-14 binding to the sigma 1 binding sites.

    Science.gov (United States)

    Nguyen, V H; Mardon, K; Kassiou, M; Christie, M D

    1999-02-01

    N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3, 10).0(5,9) .0(8,11)]dodecane (ANSTO-14) showed the highest activity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity. The present study showed that [3H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium Ki of 8.0 +/- 0.3 nM, in good agreement with the kinetic studies (Kd = 13.3 +/- 5.4 nM, n = 4), and a Bmax of 3.199 +/- 105 fmol/mg protein (n = 4). The in vivo biodistribution of [3H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with sigma ligands including (+)-pentazocine (sigma 1), ANSTO-14 (sigma 1), and DTG (sigma 1 and sigma 2) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to sigma ligands, the accumulation of [3H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [3H]ANSTO-14 is a suitable ligand for labelling sigma 1 sites in vitro but is not suitable for brain imaging of sigma binding sites in vivo.

  6. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Science.gov (United States)

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Widom, Jonathan; Wang, Xiaozhong

    2015-01-01

    The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  7. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Amy Sebeson

    Full Text Available The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  8. The binding sites for cocaine and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

    2008-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

  9. Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114

    Energy Technology Data Exchange (ETDEWEB)

    Martínez-Rodríguez, Sergio [Department of Cellular and Molecular Interactions, Vrije Universiteit Brussel, 1050 Brussels (Belgium); Ultrastructure Laboratory, Vrije Universiteit Brussel, 1050 Brussels (Belgium); González-Ramírez, Luis Antonio [Laboratorio de Estudios Cristalográficos, IACT (CSIC-U. Granada), P. T. Ciencias de la Salud, Granada 18100 (Spain); Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier [Departamento de Química Física, Bioquímica y Química Inorgánica, Área de Bioquímica y Biología Molecular, Edf. CITE I, Universidad de Almería (Spain); Gavira, Jose Antonio, E-mail: jgavira@ugr.es; García-Ruiz, Juan Ma. [Laboratorio de Estudios Cristalográficos, IACT (CSIC-U. Granada), P. T. Ciencias de la Salud, Granada 18100 (Spain); Department of Cellular and Molecular Interactions, Vrije Universiteit Brussel, 1050 Brussels (Belgium)

    2008-01-01

    Crystals of an active-site mutated hydantoin racemase from S. meliloti have been obtained in the presence and absence of d,l-5-isopropyl-hydantoin and characterized by X-ray diffraction. A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å, crystal volumes per protein weight (V{sub M}) of 1.94 and 1.98 Å{sup 3} Da{sup −1} and solvent contents of 36.7 and 37.9%, respectively.

  10. Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

    Directory of Open Access Journals (Sweden)

    Pilar Eraso

    Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

  11. Function of IHF in lambda DNA packaging. II. Effects of mutations altering the IHF binding site and the intrinsic bend in cosB on lambda development.

    Science.gov (United States)

    Xin, W; Cai, Z H; Feiss, M

    1993-03-20

    cosB is the binding site on lambda DNA for terminase, the phage DNA packaging protein. cosB contains three binding sites for gpNu1, the small subunit of terminase, and a site for integration host factor (IHF). IHF plays an accessory role in lambda DNA packaging, and IHF stimulates the burst size of lambda several-fold, presumably by assisting the interaction of terminase with cosB. The present work includes a study of the effect on lambda development of a mutation, called I1A-, which consists of three adjacent base-pair changes in the IHF binding site. The I1A- mutation was found to abolish IHF stimulation of the lambda burst size, indicating that IHF is unable to bind to the mutant I1A site. A second mutation, called I1B- and also consisting of three adjacent base-pair changes, is a mutation that reduces an intrinsic bend found in cosB. lambda I1B- was more dependent on IHF than lambda+, raising the possibility that the intrinsic bend in cosB plays a role in cos function for lambda+ under the IHF- conditions. In vitro DNA packaging experiments established that the I1 mutations affect DNA packaging per se. A series of Nu1 mutations that create terminases able to suppress a variety of cosB defects were found to suppress the defects of the I1A- and I1B- mutations under IHF- conditions.

  12. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Nielsen, Morten M.; Christiansen, Camilla

    2015-01-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch...... to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert...

  13. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  14. A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

    Science.gov (United States)

    Duffy, Caroline M; Hilbert, Brendan J; Kelch, Brian A

    2016-03-27

    The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

  15. Putative cholesterol-binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5.

    Science.gov (United States)

    Zhukovsky, Mikhail A; Lee, Po-Hsien; Ott, Albrecht; Helms, Volkhard

    2013-04-01

    Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.

  16. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers.

    Science.gov (United States)

    Farley, Emma K; Olson, Katrina M; Zhang, Wei; Rokhsar, Daniel S; Levine, Michael S

    2016-06-07

    Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.

  17. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    Directory of Open Access Journals (Sweden)

    Pene-Dumitrescu Teodora

    2012-03-01

    Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

  18. Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

    Science.gov (United States)

    Chovanec, M; Smetana, K; Purkrábková, T; Holíková, Z; Dvoránková, B; André, S; Pytlík, R; Hozák, P; Plzák, J; Sedo, A; Vacík, J; Gabius, H

    2004-01-01

    The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

  19. Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa.

    Science.gov (United States)

    Gonzalez-Guijarro, L; Lopez-Ruiz, M P; Bodegas, G; Prieto, J C; Arilla, E

    1987-04-01

    Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.

  20. Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Sigurskjold, B W; Thøgersen, H C;

    2000-01-01

    , we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find...... that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55...

  1. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Hokkaido 062-8517 (Japan)

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  2. Site-directed Chemical Modification of Recombinant Human aFGF Mutant with Polyethylene Glycol

    Institute of Scientific and Technical Information of China (English)

    Qing ZHENG; Zhi Feng HUANG; Xiao Ping WU; Zhi Jian SU; Hua XU; Wen ZHAO; Ya Dong HUANG; Xiao Kun LI

    2005-01-01

    A new product PEGylated rhaFGF was obtained by site-directed chemical modification.When compared with unmodified rhaFGF, PEGylated rhaFGF showed comparable bioactivity and superior stability at 37℃ in mouse serum and the stronger resistant potency to trypsin. This was accompanied by a substantial decreasing immunogenicity. Site-specific PEGylation of rhaFGF may increase its therapeutic potency in humans.

  3. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

  4. DNA-MATRIX: a tool for constructing transcription factor binding sites Weight matrix

    Directory of Open Access Journals (Sweden)

    Chandra Prakash Singh,

    2009-12-01

    Full Text Available Despite considerable effort to date, DNA transcription factor binding sites prediction in whole genome remains a challenge for the researchers. Currently the genome wide transcription factor binding sites prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a DNA-MATRIX tool for searching putative transcription factor binding sites in genomic sequences. DNA-MATRIX uses the simple heuristic approach for weight matrix construction, which can be transformed into different formats as per the requirement of researcher’s for further genome wide prediction and therefore provides the possibility to identify the conserved known DNA binding sites in the coregulated genes and also to search for a great variety of different regulatory binding patterns. The user may construct and save specific weight or frequency matrices in different formats derived through user selected set of known motif sequences.

  5. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli.

    Science.gov (United States)

    Chen, Lin; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-04-01

    Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.

  6. A quantitative model for the in vivo assessment of drug binding sites with positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Mintun, M.A.; Raichle, M.E.; Kilbourn, M.R.; Wooten, G.F.; Welch, M.J.

    1984-03-01

    We propose an in vivo method for use with positron emission tomography (PET) that results in a quantitative characterization of neuroleptic binding sites using radiolabeled spiperone. The data are analyzed using a mathematical model that describes transport, nonspecific binding, and specific binding in the brain. The model demonstrates that the receptor quantities Bmax (i.e., the number of binding sites) and KD-1 (i.e., the binding affinity) are not separably ascertainable with tracer methodology in human subjects. We have, therefore, introduced a new term, the binding potential, equivalent to the product BmaxKD-1, which reflects the capacity of a given tissue, or region of a tissue, for ligand-binding site interaction. The procedure for obtaining these measurements is illustrated with data from sequential PET scans of baboons after intravenous injection of carrier-added (18F)spiperone. From these data we estimate the brain tissue nonspecific binding of spiperone to be in the range of 94.2 to 95.3%, and the regional brain spiperone permeability (measured as the permeability-surface area product) to be in the range of 0.025 to 0.036 cm3/(s X ml). The binding potential of the striatum ranged from 17.4 to 21.6; these in vivo estimates compare favorably to in vitro values in the literature. To our knowledge this represents the first direct evidence that PET can be used to characterize quantitatively, locally and in vivo, drug binding sites in brain. The ability to make such measurements with PET should permit the detailed investigation of diseases thought to result from disorders of receptor function.

  7. Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21).

    Science.gov (United States)

    Young, Kendra A; Chen, Xiaojiang S; Holers, V Michael; Hannan, Jonathan P

    2007-12-14

    Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.

  8. Ivermectin binding sites in human and invertebrate Cys-loop receptors

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2012-01-01

    Ivermectin is a gold standard antiparasitic drug that has been used successfully to treat billions of humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular mo...... for a wide variety of human neurological disorders....

  9. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

    Directory of Open Access Journals (Sweden)

    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  10. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation.

    Science.gov (United States)

    Quick, Matthias; Winther, Anne-Marie Lund; Shi, Lei; Nissen, Poul; Weinstein, Harel; Javitch, Jonathan A

    2009-04-07

    The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na(+); later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule approximately 11 A above the bound leucine and 2 Na(+). We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na(+)-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-beta-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 A the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na(+)-coupled symport.

  11. Discriminating between HuR and TTP binding sites using the k-spectrum kernel method

    Science.gov (United States)

    Goldberg, Debra S.; Dowell, Robin

    2017-01-01

    Background The RNA binding proteins (RBPs) human antigen R (HuR) and Tristetraprolin (TTP) are known to exhibit competitive binding but have opposing effects on the bound messenger RNA (mRNA). How cells discriminate between the two proteins is an interesting problem. Machine learning approaches, such as support vector machines (SVMs), may be useful in the identification of discriminative features. However, this method has yet to be applied to studies of RNA binding protein motifs. Results Applying the k-spectrum kernel to a support vector machine (SVM), we first verified the published binding sites of both HuR and TTP. Additional feature engineering highlighted the U-rich binding preference of HuR and AU-rich binding preference for TTP. Domain adaptation along with multi-task learning was used to predict the common binding sites. Conclusion The distinction between HuR and TTP binding appears to be subtle content features. HuR prefers strongly U-rich sequences whereas TTP prefers AU-rich as with increasing A content, the sequences are more likely to be bound only by TTP. Our model is consistent with competitive binding of the two proteins, particularly at intermediate AU-balanced sequences. This suggests that fine changes in the A/U balance within a untranslated region (UTR) can alter the binding and subsequent stability of the message. Both feature engineering and domain adaptation emphasized the extent to which these proteins recognize similar general sequence features. This work suggests that the k-spectrum kernel method could be useful when studying RNA binding proteins and domain adaptation techniques such as feature augmentation could be employed particularly when examining RBPs with similar binding preferences. PMID:28333956

  12. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    Science.gov (United States)

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  13. Characterization of two different melatonin binding sites in peripheral tissues of the teleost Tinca tinca.

    Science.gov (United States)

    López Patiño, M A; Guijarro, A I; Alonso-Gómez, A L; Delgado, M J

    2012-01-01

    The aim of the present study was to localize and characterize 2-iodo-melatonin ([(125)I]Mel) binding sites in peripheral tissues of the teleost Tinca tinca. A wide distribution of [(125)I]Mel binding sites in peripheral locations of the tench is found, with highest densities being measured in the heart, gills and kidney, and low density of [(125)I]Mel binding sites in gastrointestinal tract, spleen, liver and gonads. Saturation, kinetics, and pharmacological approaches revealed the presence of, at least, two different [(125)I]Mel binding sites in the tench peripheral tissues. The unique characterized subtype in the heart fulfils all the criteria for a canonical melatonin receptor belonging to MT(1) family (the binding is saturable, reversible, and inhibited by GTP analogs), and gives support for the presence of a functional melatonin receptor in the heart of the tench. In contrast, kinetic and pharmacological studies in the kidney revealed the preponderance of a melatonin binding site belonging to the MT(3)-like receptor subtype. Moreover, the decrease of specific binding in both, heart and kidney membranes, and the decrease of affinity in the kidney, produced by the addition of a non-hydrolysable GTP analog, and sodium cations suggest the presence of G(i/o)-proteins (that mediate inhibition of cAMP formation) coupled to such melatonin binding sites. Our results also point to different G(i/o)-proteins involved in the underlying mechanism of melatonin binding sites activation in the kidney. Additionally, the kinetics of [(125)I]Mel binding in kidney membrane preparations is a highly thermosensitive process, being necessary to perform the assays at 4 °C since the equilibrium was not reached at 25 °C assay temperature. The time needed to complete association of [(125)I]Mel at such low temperature is only 15s, whereas 100s is required to displace [(125)I]Mel specific binding by the unlabeled melatonin in kidney membranes. Present results support previous reports on

  14. PhyloScan: identification of transcription factor binding sites using cross-species evidence

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    Newberg Lee A

    2007-01-01

    Full Text Available Abstract Background When transcription factor binding sites are known for a particular transcription factor, it is possible to construct a motif model that can be used to scan sequences for additional sites. However, few statistically significant sites are revealed when a transcription factor binding site motif model is used to scan a genome-scale database. Methods We have developed a scanning algorithm, PhyloScan, which combines evidence from matching sites found in orthologous data from several related species with evidence from multiple sites within an intergenic region, to better detect regulons. The orthologous sequence data may be multiply aligned, unaligned, or a combination of aligned and unaligned. In aligned data, PhyloScan statistically accounts for the phylogenetic dependence of the species contributing data to the alignment and, in unaligned data, the evidence for sites is combined assuming phylogenetic independence of the species. The statistical significance of the gene predictions is calculated directly, without employing training sets. Results In a test of our methodology on synthetic data modeled on seven Enterobacteriales, four Vibrionales, and three Pasteurellales species, PhyloScan produces better sensitivity and specificity than MONKEY, an advanced scanning approach that also searches a genome for transcription factor binding sites using phylogenetic information. The application of the algorithm to real sequence data from seven Enterobacteriales species identifies novel Crp and PurR transcription factor binding sites, thus providing several new potential sites for these transcription factors. These sites enable targeted experimental validation and thus further delineation of the Crp and PurR regulons in E. coli. Conclusion Better sensitivity and specificity can be achieved through a combination of (1 using mixed alignable and non-alignable sequence data and (2 combining evidence from multiple sites within an intergenic

  15. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  16. Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

    Science.gov (United States)

    Husu, Ivan; Magyar, Melinda; Szabó, Tibor; Fiser, Béla; Gómez-Bengoa, Enrique; Nagy, László

    2015-04-01

    Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

  17. Membrane androgen binding sites are preferentially expressed in human prostate carcinoma cells

    Directory of Open Access Journals (Sweden)

    Delakas Dimitrios

    2003-01-01

    Full Text Available Abstract Background Prostate cancer is one of the most frequent malignancies in males. Nevertheless, to this moment, there is no specific routine diagnostic marker to be used in clinical practice. Recently, the identification of a membrane testosterone binding site involved in the remodeling of actin cytoskeleton structures and PSA secretion, on LNCaP human prostate cancer cells has been reported. We have investigated whether this membrane testosterone binding component could be of value for the identification of prostate cancer. Methods Using a non-internalizable testosterone-BSA-FITC analog, proven to bind on membrane sites only in LNCaP cells, we have investigated the expression of membrane testosterone binding sites in a series of prostate carcinomas (n = 14, morphologically normal epithelia, taken from areas of the surgical specimens far from the location of the carcinomas (n = 8 and benign prostate hyperplasia epithelia (n = 10. Isolated epithelial cells were studied by flow cytometry, and touching preparations, after 10-min incubation. In addition, routine histological slides were assayed by confocal laser microscopy. Results We show that membrane testosterone binding sites are preferentially expressed in prostate carcinoma cells, while BPH and non-malignant epithelial cells show a low or absent binding. Conclusions Our results indicate that membrane testosterone receptors might be of use for the rapid routine identification of prostate cancer, representing a new diagnostic marker of the disease.

  18. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  19. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

  20. Autoradiographic demonstration of oxytocin-binding sites in the macula densa

    Energy Technology Data Exchange (ETDEWEB)

    Stoeckel, M.E.; Freund-Mercier, M.J. (Centre National de la Recherche Scientifique, Strasbourg (France))

    1989-08-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective {sup 125}I-labeled OT antagonist ({sup 125}I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of {sup 125}I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that {sup 125}I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration.

  1. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  2. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    Science.gov (United States)

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

  3. Functional identification of catalytic metal ion binding sites within RNA.

    Directory of Open Access Journals (Sweden)

    James L Hougland

    2005-09-01

    Full Text Available The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s. In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

  4. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

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    John A Capra

    2009-12-01

    Full Text Available Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/.

  5. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    Science.gov (United States)

    Capra, John A; Laskowski, Roman A; Thornton, Janet M; Singh, Mona; Funkhouser, Thomas A

    2009-12-01

    Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

  6. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  7. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  8. Binding Sites of miR-1273 Family on the mRNA of Target Genes

    Directory of Open Access Journals (Sweden)

    Anatoly Ivashchenko

    2014-01-01

    Full Text Available This study examined binding sites of 2,578 miRNAs in the mRNAs of 12,175 human genes using the MirTarget program. It found that the miRNAs of miR-1273 family have between 33 and 1,074 mRNA target genes, with a free hybridization energy of 90% or more of its maximum value. The miR-1273 family consists of miR-1273a, miR-1273c, miR-1273d, miR-1273e, miR-1273f, miR-1273g-3p, miR-1273g-5p, miR-1273h-3p, and miR-1273h-5p. Unique miRNAs (miR-1273e, miR-1273f, and miR-1273g-3p have more than 400 target genes. We established 99 mRNA nucleotide sequences that contain arranged binding sites for the miR-1273 family. High conservation of each miRNA binding site in the mRNA of the target genes was found. The arranged binding sites of the miR-1273 family are located in the 5′UTR, CDS, or 3′UTR of many mRNAs. Five repeating sites containing some of the miR-1273 family’s binding sites were found in the 3′UTR of several target genes. The oligonucleotide sequences of miR-1273 binding sites located in CDSs code for homologous amino acid sequences in the proteins of target genes. The biological role of unique miRNAs was also discussed.

  9. Effects of sodium on cell surface and intracellular TH-naloxone binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Pollack, A.E.; Wooten, G.F.

    1987-07-27

    The binding of the opiate antagonist TH-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, TH-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables.

  10. Prenatal exposure to methylmercury alters development of adrenergic receptor binding sites in peripheral sympathetic target tissues

    Energy Technology Data Exchange (ETDEWEB)

    Slotkin, T.A.; Orband, L.; Cowdery, T.; Kavlock, R.J.; Bartolome, J.

    1987-01-01

    In order to assess the impact of prenatal exposure to methylmercury on sympathetic neurotransmission, effects on development of adrenergic receptor binding sites in peripheral tissues was evaluated. In the liver, methylmercury produced a dose-dependent increase in alpha/sub 1/, alpha/sub 2/, and beta-receptor binding of radioliganda throughout the first 5 weeks of postnatal life. Similarly, renal alpha-receptor subtypes showed increased binding capabilities, but binding to alpha-receptor sites was reduced. At least some of the changes in receptors appear to be of functional significance, as physiological reactivity to adrenergic stimulation is altered in the same directions in these two tissues. The actions of methylmercury displayed tissue specificity in that the same receptor populations were largely unaffected in other tissues (lung, heart). These results suggest that methylmercury exposure in utero alters adrenergic responses through targeted effects on postsynaptic receptor populations in specific tissues.

  11. Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

    Directory of Open Access Journals (Sweden)

    Lim Carmay

    2007-03-01

    Full Text Available Abstract Background For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg2+, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg2+-proteins share insufficient sequence homology to identify Mg2+-specific sequence motifs, they may still share similarity in the Mg2+-binding site structure. However, no structural motifs characteristic of Mg2+-binding sites have been reported. Thus, our aims are (i to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii to apply it to Mg2+-proteins sharing 2+-structural motifs are identified as recurring structural patterns. Results The structural alphabet-based motif discovery method has revealed the structural preference of Mg2+-binding sites for certain local/secondary structures: compared to all residues in the Mg2+-proteins, both first and second-shell Mg2+-ligands prefer loops to helices. Even when the Mg2+-proteins share no significant sequence homology, some of them share a similar Mg2+-binding site structure: 4 Mg2+-structural motifs, comprising 21% of the binding sites, were found. In particular, one of the Mg2+-structural motifs found maps to a specific functional group, namely, hydrolases. Furthermore, 2 of the motifs were not found in non metalloproteins or in Ca2+-binding proteins. The structural motifs discovered thus capture some essential biochemical and/or evolutionary properties, and hence may be useful for discovering proteins where Mg2+ plays an important biological role. Conclusion The structural motif discovery method presented herein is general and can be applied to any set of proteins with known 3D structures. This new method is timely considering the increasing number of structures for

  12. Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of Enterotoxigenic Escherichia coli and CfaE-R181A mutant

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    Iram Liaqat

    2012-09-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2. Further, enzyme-linked immunosorbent assay (ELISA assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3 and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacIq/∆cotD (CS2 strain and MC4100/pEU2124 (CfaE-R181A mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacIq/pEU5881 showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

  13. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    Science.gov (United States)

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  14. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    Science.gov (United States)

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-07-23

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

  15. Deep sequencing of MYC DNA-binding sites in Burkitt lymphoma.

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    Volkhard Seitz

    Full Text Available BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP followed by next generation sequencing (ChIP-Seq has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i many B-cell relevant genes are targeted by MYC and (ii that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.

  16. An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

    Science.gov (United States)

    Schanke, J T; Marcuzzi, A; Podzorski, R P; Van Ness, B

    1994-01-01

    Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells. Images PMID:7816634

  17. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

    Science.gov (United States)

    Sandhya, B; Hegde, Ashwini H; Kalanur, Shankara S; Katrahalli, Umesha; Seetharamappa, J

    2011-04-01

    The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Förster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.

  18. Marked reduction in the number of platelet-tritiated imipramine binding sites in geriatric depression

    Energy Technology Data Exchange (ETDEWEB)

    Nemeroff, C.B.; Knight, D.L.; Krishnan, R.R.; Slotkin, T.A.; Bissette, G.; Melville, M.L.; Blazer, D.G.

    1988-10-01

    The number (Bmax) and affinity (Kd) of platelet-tritiated imipramine binding sites was determined in young and middle-aged controls 50 years of age and younger (n = 25), elderly normal controls over 60 years of age (n = 18), patients who fulfilled DSM-III criteria for major depression who were under 50 years of age (n = 29), patients who fulfilled DSM-III criteria for major depression who were 60 years of age and older (n = 19), and patients who fulfilled both DSM-III criteria for primary degenerative dementia and National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable Alzheimer's disease (n = 13). Both groups of depressed patients (under 50 and over 60 years of age) exhibited significant reductions (decreases 42%) in the number of platelet-tritiated imipramine binding sites with no change in affinity, when compared with their age-matched controls. There was little overlap in Bmax values between the elderly depressed patients and their controls. The patients with probable Alzheimer's disease showed no alteration in platelet-tritiated imipramine binding. There was no statistically significant relationship between postdexamethasone plasma cortisol concentrations and tritiated imipramine binding. These results indicate that platelet-tritiated imipramine binding may have potential utility as a diagnostic adjunct in geriatric depression, and moreover that the reduction in the number of platelet-tritiated imipramine binding sites is not due to hypercortisolemia.

  19. Functional diversification of paralogous transcription factors via divergence in DNA binding site motif and in expression.

    Directory of Open Access Journals (Sweden)

    Larry N Singh

    Full Text Available BACKGROUND: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. FINDINGS: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. CONCLUSIONS: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression

  20. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

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    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  1. Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    Institute of Scientific and Technical Information of China (English)

    Manali; Phadke; Natalia; Krynetskaia; Anurag; Mishra; Carlos; Barrero; Salim; Merali; Scott; A; Gothe; Evgeny; Krynetskiy

    2015-01-01

    AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.

  2. Autoradiographic distribution of /sup 125/I-galanin binding sites in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Skofitsch, G.; Sills, M.A.; Jacobowitz, D.M.

    1986-11-01

    Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of /sup 125/I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. /sup 125/I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H1 and H2, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.

  3. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators.

    Science.gov (United States)

    Karapetyan, Sargis; Buchler, Nicolas E

    2015-12-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

  4. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    Science.gov (United States)

    Karapetyan, Sargis; Buchler, Nicolas E.

    2015-12-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

  5. Cortisol decreases 2[[sup 125]I] iodomelatonin binding sites in the duck thymus

    Energy Technology Data Exchange (ETDEWEB)

    Poon, A.M.S.; Liu, Z.M.; Tang, F.; Pang, S.F. (Univ. of Hong Kong (China))

    1994-03-01

    The immunosuppressive effect of chronic glucocorticoid treatment on 2[[sup 125]I] iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for seven days. 2[[sup 125]I] iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[[sup 125]I] iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[[sup 125]I] iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. The authors findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested. 34 refs., 3 tabs.

  6. The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase.

    Science.gov (United States)

    Fitzgerald, M M; Trester, M L; Jensen, G M; McRee, D E; Goodin, D B

    1995-09-01

    The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.

  7. Identification of a functional hepatocyte nuclear factor 4 binding site in the neutral ceramidase promoter

    DEFF Research Database (Denmark)

    Maltesen, Henrik R; Troelsen, Jesper T; Olsen, Jørgen

    2010-01-01

    in ceramide digestion. It was the purpose of the present work to experimentally verify the functional importance of a HNF-4a binding site predicted by bioinformatic analysis to be present in the Asah2 promoter. Using supershift analysis, HNF-4a overexpression, and HNF-4a knockdown experiments it was confirmed...... that the predicted HNF-4a binding site identified in the Asah2 promoter is functional. The results support the hypothesis that HNF-4a might be important for intestinal glycolipid metabolism....

  8. Does distant homology with Evf reveal a lipid binding site in Bacillus thuringiensis cytolytic toxins?

    Science.gov (United States)

    Rigden, Daniel J

    2009-05-19

    The Cry and Cyt classes of insecticidal toxins derived from the sporulating bacterium Bacillus thuringiensis are valuable substitutes for synthetic pesticides in agricultural contexts. Crystal structures and many biochemical data have provided insights into their molecular mechanisms, generally thought to involve oligomerization and pore formation, but have not localised the site on Cyt toxins responsible for selective binding of phospholipids containing unsaturated fatty acids. Here, distant homology between the structure of Cyt toxins and Erwinia virulence factor (Evf) is demonstrated which, along with sequence conservation analysis, allows a putative lipid binding site to be localised in the toxins.

  9. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo;

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor......The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly...

  10. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites

    OpenAIRE

    Zahid, Henna; Miah, Layeque; Lau, Andy; Brochard, Lea; Hati, Debolina; Bui, T. T.; Drake, A. F.; Gor, Jayesh; Perkins, Stephen J.; McDermott, Lindsay C.

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigate...

  11. Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

    DEFF Research Database (Denmark)

    Møller, Charlotte; Sprenger, Richard R; Stürup, Stefan;

    2011-01-01

    and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges...... were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached...

  12. Rational design of a protein that binds integrin αvβ3 outside the ligand binding site

    Science.gov (United States)

    Turaga, Ravi Chakra; Yin, Lu; Yang, Jenny J.; Lee, Hsiauwei; Ivanov, Ivaylo; Yan, Chunli; Yang, Hua; Grossniklaus, Hans E.; Wang, Siming; Ma, Cheng; Sun, Li; Liu, Zhi-Ren

    2016-01-01

    Integrin αvβ3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αvβ3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics. PMID:27241473

  13. Rational design of a protein that binds integrin αvβ3 outside the ligand binding site.

    Science.gov (United States)

    Turaga, Ravi Chakra; Yin, Lu; Yang, Jenny J; Lee, Hsiauwei; Ivanov, Ivaylo; Yan, Chunli; Yang, Hua; Grossniklaus, Hans E; Wang, Siming; Ma, Cheng; Sun, Li; Liu, Zhi-Ren

    2016-05-31

    Integrin αvβ3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αvβ3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics.

  14. Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin.

    Science.gov (United States)

    Wang, C L; Leavis, P C; Gergely, J

    1984-12-18

    The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.

  15. Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces

    DEFF Research Database (Denmark)

    Sauer, Jørgen; Abou Hachem, Maher; Svensson, Birte;

    2013-01-01

    We here report a quantitative study on the binding kinetics of inhibition of the enzyme glucoamylase and how individual active site amino acid mutations influence kinetics. To address this challenge, we have developed a fast and efficient method for anchoring native acarbose to gold chip surfaces...... shown that at pH 7.0 the association and dissociation rate constants for the acarbose-glucoamylase interaction are 104M−1s−1 and 103s−1, respectively, and that the conformational change to a tight enzyme–inhibitor complex affects the dissociation rate constant by a factor of 102s−1. Additionally...

  16. Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase.

    Directory of Open Access Journals (Sweden)

    Aleksandra Usenik

    Full Text Available As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1 level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.

  17. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W. (Toronto); (UBC)

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  18. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

    OpenAIRE

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-01-01

    Experiments are presented which suggest that the binding of the primer tRNA to the primer binding site of the HIV-1 5′ UTR is involved in the dimerization of the genome, as part of the packaging process.

  19. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis.

    Science.gov (United States)

    Wang, Qishan; Bag, Jnanankur

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including alpha-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  20. The importance of relative mutant level for evaluating impact on outcome of KIT, FLT3 and CBL mutations in core-binding factor acute myeloid leukemia.

    Science.gov (United States)

    Allen, C; Hills, R K; Lamb, K; Evans, C; Tinsley, S; Sellar, R; O'Brien, M; Yin, J L; Burnett, A K; Linch, D C; Gale, R E

    2013-09-01

    Several different mutations collaborate with the fusion proteins in core-binding factor acute myeloid leukemia (CBF-AML) to induce leukemogenesis, but their prognostic significance remains unclear. We screened 354 predominantly younger (mutations and FLT3 internal tandem duplications (FLT3(ITD)) and assessed the impact of relative mutant level on outcome. Overall, 28% had KIT, 6% FLT3(ITD), 10% FLT3(TKD), 27% RAS and 6% CBL mutations. Mutant levels for all genes/loci were highly variable. KIT mutations were associated with a higher cumulative incidence of relapse but in multivariate analysis this was only significant for cases with a higher mutant level of 25% or greater (95% confidence interval (CI)=1.01-1.52, P=0.04). Similarly, only FLT3(ITD-HIGH) was a significant adverse factor for overall survival (OS; CI=1.27-5.39, P=0.004). Conversely, FLT3(TKD-HIGH) and CBL(HIGH) were both favorable factors for OS (CI= 0.31-0.89, P=0.01 and CI=0.05-0.85, P=0.02, respectively). KIT mutations were frequently lost at relapse, which is relevant to minimal residual disease detection and the clinical use of KIT inhibitors. These results indicate that relative mutant level should be taken into account when evaluating the impact of mutations in CBF-AML.

  1. A specific binding site recognizing a fragment of angiotensin II in bovine adrenal cortex membranes.

    Science.gov (United States)

    Bernier, S G; Fournier, A; Guillemette, G

    1994-12-12

    We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.24 +/- 0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37 degrees C revealed a calculated kinetic Kd of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT1 or AT2. Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3-7) (Val-Tyr-Ile-His-Pro) > angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) > or = angiotensin II-(4-7) (Tyr-Ile-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > angiotensin II-(1-6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4-8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3-6) (Val-Tyr-Ile-His), angiotensin II-(4-6) (Tyr-Ile-His), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'(1-H-tetrazol-5-yl)[1,1'-biphenyl]-4-y l) methyl]-3-H-imidazo[4,5-beta]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)4,5,6 ,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensioffn IV to bovine adrenal cortex membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Identification and characterization of a glycosaminoglycan binding site on interleukin-10 via molecular simulation methods.

    Science.gov (United States)

    Gehrcke, Jan-Philip; Pisabarro, M Teresa

    2015-11-01

    The biological function of the pleiotropic cytokine interleukin-10 (IL-10), which has an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are essential constituents of the extracellular matrix with an important role in modulating the biological function of many proteins. The molecular mechanisms governing the IL-10-GAG interaction, though, are unclear so far. In particular, detailed knowledge about GAG binding sites and recognition mode on IL-10 is lacking, despite of its imminent importance for understanding the functional consequences of IL-10-GAG interaction. In the present work, we report a GAG binding site on IL-10 identified by applying computational methods based on Coulomb potential calculations and specialized molecular dynamics simulations. The identified GAG binding site is constituted of several positively charged residues, which are conserved among species. Exhaustive conformational space sampling of a series of GAG ligands binding to IL-10 led to the observation of two GAG binding modes in the predicted binding site, and to the identification of IL-10 residues R104, R106, R107, and K119 as being most important for molecular GAG recognition. In silico mutation as well as single-residue energy decomposition and detailed analysis of hydrogen-bonding behavior led to the conclusion that R107 is most essential and assumes a unique role in IL-10-GAG interaction. This structural and dynamic characterization of GAG-binding to IL-10 represents an important step for further understanding the modulation of the biological function of IL-10.

  3. Identification of Calcium binding sites on calsequestrin 1 and its implications to polymerization

    Science.gov (United States)

    Kumar, Amit; Chakravarty, Harapriya; Bal, Naresh C.; Balaraju, Tuniki; Jena, Nivedita; Misra, Gauri; Bal, Chandralata; Pieroni, Enrico; Periasamy, Muthu; Sharon, Ashoke

    2013-01-01

    Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70–80 Ca2+ ions. However, the nature of Ca2+-binding sites has not yet been fully characterized. In this study, we employed in-silico approaches to identify the Ca2+ binding sites and to understand the molecular basis of CASQ1-Ca2+ recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: 3UOM) and adding the missing C-terminal residues (aa350–364). Using this model we performed extensive 30 ns molecular dynamics simulations exposed to wide range of Ca2+ concentrations ([Ca2+]). Our results show that the Ca2+-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca2+-binding sites share a similar geometry and interestingly, majority of them were found to be induced by increased [Ca2+]. We also found that the system undergoes maximal Ca2+-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data shows that the CASQ1 back-to-back stacking is progressively stabilized by emergence of an increasing number of hydrophobic interactions with increasing [Ca2+]. Further, this study shows that the CAS domain assumes a compact structure with increase in Ca2+ binding, which suggests that the CAS domain might function as a Ca2+-sensor that may be a novel structural motif to sense metal. We propose the term “Dn-motif” for the CAS domain. PMID:23629537

  4. The phenotype of many independently isolated +1 frameshift suppressor mutants supports a pivotal role of the P-site in reading frame maintenance.

    Science.gov (United States)

    Jäger, Gunilla; Nilsson, Kristina; Björk, Glenn R

    2013-01-01

    The main features of translation are similar in all organisms on this planet and one important feature of it is the way the ribosome maintain the reading frame. We have earlier characterized several bacterial mutants defective in tRNA maturation and found that some of them correct a +1 frameshift mutation; i.e. such mutants possess an error in reading frame maintenance. Based on the analysis of the frameshifting phenotype of such mutants we proposed a pivotal role of the ribosomal grip of the peptidyl-tRNA to maintain the correct reading frame. To test the model in an unbiased way we first isolated many (467) independent mutants able to correct a +1 frameshift mutation and thereafter tested whether or not their frameshifting phenotypes were consistent with the model. These 467+1 frameshift suppressor mutants had alterations in 16 different loci of which 15 induced a defective tRNA by hypo- or hypermodifications or altering its primary sequence. All these alterations of tRNAs induce a frameshift error in the P-site to correct a +1 frameshift mutation consistent with the proposed model. Modifications next to and 3' of the anticodon (position 37), like 1-methylguanosine, are important for proper reading frame maintenance due to their interactions with components of the ribosomal P-site. Interestingly, two mutants had a defect in a locus (rpsI), which encodes ribosomal protein S9. The C-terminal of this protein contacts position 32-34 of the peptidyl-tRNA and is thus part of the P-site environment. The two rpsI mutants had a C-terminal truncated ribosomal protein S9 that destroys its interaction with the peptidyl-tRNA resulting in +1 shift in the reading frame. The isolation and characterization of the S9 mutants gave strong support of our model that the ribosomal grip of the peptidyl-tRNA is pivotal for the reading frame maintenance.

  5. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  6. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  7. Binding isotope effects as a tool for distinguishing hydrophobic and hydrophilic binding sites of HIV-1 RT.

    Science.gov (United States)

    Krzemińska, Agnieszka; Paneth, Piotr; Moliner, Vicent; Świderek, Katarzyna

    2015-01-22

    The current treatment for HIV-1 infected patients consists of a cocktail of inhibitors, in an attempt to improve the potency of the drugs by adding the possible effects of each supplied compound. In this contribution, nine different inhibitors of HIV-1 RT, one of the three key proteins responsible for the virus replication, have been selected to develop and test a computational protocol that allows getting a deep insight into the inhibitors' binding mechanism. The interaction between the inhibitors and the protein have been quantified by computing binding free energies through FEP calculations, while a more detailed characterization of the kind of inhibitor-protein interactions is based on frequency analysis of the ligands in the initial and final state, i.e. in solution and binding the protein. QM/MM calculation of heavy atoms ((13)C, (15)N, and (18)O) binding isotope effects (BIE) have been used to identify the binding sites of the different inhibitors. Specific interactions between the isotopically labeled atoms of the inhibitors and polar residues and magnesium cations on the hydrophilic pocket of the protein are responsible for the frequencies shifting that can be detected when comparing the IR spectra of the compounds in solution and in the protein. On the contrary, it seems that changes in vdW interactions from solution to the final state when the ligand is interacting with residues of the hydrophobic cavity, does not influence frequency modes and then no BIE are observed. Our results suggest that a proper computational protocol can be a valuable tool which in turn can be used to increase the efficiency of anti AIDS drugs.

  8. A reexamination of information theory-based methods for DNA-binding site identification

    Directory of Open Access Journals (Sweden)

    O'Neill Michael C

    2009-02-01

    Full Text Available Abstract Background Searching for transcription factor binding sites in genome sequences is still an open problem in bioinformatics. Despite substantial progress, search methods based on information theory remain a standard in the field, even though the full validity of their underlying assumptions has only been tested in artificial settings. Here we use newly available data on transcription factors from different bacterial genomes to make a more thorough assessment of information theory-based search methods. Results Our results reveal that conventional benchmarking against artificial sequence data leads frequently to overestimation of search efficiency. In addition, we find that sequence information by itself is often inadequate and therefore must be complemented by other cues, such as curvature, in real genomes. Furthermore, results on skewed genomes show that methods integrating skew information, such as Relative Entropy, are not effective because their assumptions may not hold in real genomes. The evidence suggests that binding sites tend to evolve towards genomic skew, rather than against it, and to maintain their information content through increased conservation. Based on these results, we identify several misconceptions on information theory as applied to binding sites, such as negative entropy, and we propose a revised paradigm to explain the observed results. Conclusion We conclude that, among information theory-based methods, the most unassuming search methods perform, on average, better than any other alternatives, since heuristic corrections to these methods are prone to fail when working on real data. A reexamination of information content in binding sites reveals that information content is a compound measure of search and binding affinity requirements, a fact that has important repercussions for our understanding of binding site evolution.

  9. A reexamination of information theory-based methods for DNA-binding site identification

    Science.gov (United States)

    Erill, Ivan; O'Neill, Michael C

    2009-01-01

    Background Searching for transcription factor binding sites in genome sequences is still an open problem in bioinformatics. Despite substantial progress, search methods based on information theory remain a standard in the field, even though the full validity of their underlying assumptions has only been tested in artificial settings. Here we use newly available data on transcription factors from different bacterial genomes to make a more thorough assessment of information theory-based search methods. Results Our results reveal that conventional benchmarking against artificial sequence data leads frequently to overestimation of search efficiency. In addition, we find that sequence information by itself is often inadequate and therefore must be complemented by other cues, such as curvature, in real genomes. Furthermore, results on skewed genomes show that methods integrating skew information, such as Relative Entropy, are not effective because their assumptions may not hold in real genomes. The evidence suggests that binding sites tend to evolve towards genomic skew, rather than against it, and to maintain their information content through increased conservation. Based on these results, we identify several misconceptions on information theory as applied to binding sites, such as negative entropy, and we propose a revised paradigm to explain the observed results. Conclusion We conclude that, among information theory-based methods, the most unassuming search methods perform, on average, better than any other alternatives, since heuristic corrections to these methods are prone to fail when working on real data. A reexamination of information content in binding sites reveals that information content is a compound measure of search and binding affinity requirements, a fact that has important repercussions for our understanding of binding site evolution. PMID:19210776

  10. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  11. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites

    OpenAIRE

    Sena, J.A.D. [UNESP; Hernández Rodríguez, Carmen Sara; Ferré Manzanero, Juan

    2009-01-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  12. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites.

    Science.gov (United States)

    Sena, Janete A D; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-04-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  13. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  14. Functional roles of the non-catalytic calcium-binding sites in the N-terminal domain of human peptidylarginine deiminase 4.

    Science.gov (United States)

    Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

    2013-01-01

    This study investigated the functional roles of the N-terminal Ca(2+) ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca(2+)-binding site of PAD4 were mutated to disrupt the binding of Ca(2+) ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat)/K(m,BAEE) values were 0.02, 0.63 and 0.01 s(-1)mM(-1) (20.8 s(-1)mM(-1) for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat) value of 0.3 s(-1) (13.3 s(-1) for wild-type), whereas D176A retained some catalytic power with a k(cat) of 9.7 s(-1). Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat)/K(m,BAEE) values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+) indicated that the conformational stability of the enzyme is highly dependent on Ca(2+) ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+) ions in the N-terminal Ca(2+)-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+) ions play critical roles in the full activation of the PAD4 enzyme.

  15. Functional roles of the non-catalytic calcium-binding sites in the N-terminal domain of human peptidylarginine deiminase 4.

    Directory of Open Access Journals (Sweden)

    Yi-Liang Liu

    Full Text Available This study investigated the functional roles of the N-terminal Ca(2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4. Amino acid residues located in the N-terminal Ca(2+-binding site of PAD4 were mutated to disrupt the binding of Ca(2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat/K(m,BAEE values were 0.02, 0.63 and 0.01 s(-1mM(-1 (20.8 s(-1mM(-1 for WT, respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat value of 0.3 s(-1 (13.3 s(-1 for wild-type, whereas D176A retained some catalytic power with a k(cat of 9.7 s(-1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat/K(m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+ indicated that the conformational stability of the enzyme is highly dependent on Ca(2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+ ions in the N-terminal Ca(2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+ ions play critical roles in the full activation of the PAD4 enzyme.

  16. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.;

    2002-01-01

    The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  17. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  18. Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy.

    Science.gov (United States)

    Matsubara, Teruhiko; Onishi, Ai; Yamaguchi, Daisuke; Sato, Toshinori

    2016-03-01

    The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar-protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

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

  20. Mapping cocaine binding sites in human and baboon brain in vivo.

    Science.gov (United States)

    Fowler, J S; Volkow, N D; Wolf, A P; Dewey, S L; Schlyer, D J; Macgregor, R R; Hitzemann, R; Logan, J; Bendriem, B; Gatley, S J

    1989-01-01

    The first direct measurements of cocaine binding in the brain of normal human volunteers and baboons have been made by using positron emission tomography (PET) and tracer doses of [N-11C-methyl]-(-)-cocaine ([11C]cocaine). Cocaine's binding and release from brain are rapid with the highest regional uptake of carbon-11 occurring in the corpus striatum at 4-10 minutes after intravenous injection of labeled cocaine. This was followed by a clearance to half the peak value at about 25 minutes with the overall time course paralleling the previously documented time course of the euphoria experienced after intravenous cocaine administration. Blockade of the dopamine reuptake sites with nomifensine reduced the striatal but not the cerebellar uptake of [11C]cocaine in baboons indicating that cocaine binding is associated with the dopamine reuptake site in the corpus striatum. A comparison of labeled metabolites of cocaine in human and baboon plasma showed that while cocaine is rapidly metabolized in both species, the profile of labeled metabolites is different, with baboon plasma containing significant amounts of labeled carbon dioxide, and human plasma containing no significant labeled carbon dioxide. These studies demonstrate the feasibility of using [11C]cocaine and PET to map binding sites for cocaine in human brain, to monitor its kinetics, and to characterize its binding mechanism by using appropriate pharmacological challenges.

  1. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas;

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  2. Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

    Directory of Open Access Journals (Sweden)

    Previato Jose O

    2008-05-01

    Full Text Available Abstract Background The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs. In the present study, nuclear magnetic resonance (NMR was used to map the binding site(s of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. Results The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. Conclusion Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

  3. Mapping the heparin-binding site on the 13-14F3 fragment of fibronectin.

    Science.gov (United States)

    Sachchidanand; Lequin, Olivier; Staunton, David; Mulloy, Barbara; Forster, Mark J; Yoshida, Keiichi; Campbell, Iain D

    2002-12-27

    Fibronectin, a multifunctional glycoprotein of the extracellular matrix, plays a major role in cell adhesion. Various studies have revealed that the human 13th and 14th fibronectin type III domains (labeled (13)F3 and (14)F3 here) contain a heparin-binding site. Mapping of the heparin-binding sites of (13-14)F3, (13)F3, and (14)F3 by NMR chemical shift perturbation, isothermal titration calorimetry, and molecular modeling show that (13)F3 provides the dominant heparin-binding site and that the residues involved are within the first 29 amino acids of (13)F3. Predictions from earlier biochemical and modeling studies as well as the x-ray structure of (12-14)F3 were tested. It was shown that the positively charged residues that project into the solvent from the ABE face of the triple-stranded beta sheet on (13)F3 are involved in binding, but (14)F3 does not appear to contribute significantly to heparin binding.

  4. Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Saavedra, J.M.; Israel, A.; Plunkett, L.M.; Kurihara, M.; Shigematsu, K.; Correa, F.M.

    1986-07-01

    Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM /sup 125/I-(Sar1)-angiotensin II, (/sup 3/H)-Ultrofilm autoradiography, computerized microdensitometry and comparison with /sup 125/I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with /sup 125/I-(Sar1)-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.

  5. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  6. Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis.

    Directory of Open Access Journals (Sweden)

    Simon Oakley

    Full Text Available Barbiturates potentiate GABA actions at the GABA(A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10-500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

  7. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    Directory of Open Access Journals (Sweden)

    Zhichao Miao

    2015-12-01

    Full Text Available Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i the definition of nucleic acid binding sites; ii the training and test datasets; iii the algorithmic methods for the prediction strategies; iv the performance measures and v the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy… are applied. We found that i the tools have been greatly improved over the years; ii some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii RNA binding and DNA binding appear to follow similar driving forces and iv dataset bias may exist in some methods.

  8. Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

  9. Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

    OpenAIRE

    Rebelo, Tânia S. C. R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M. H.; Noronha, João P. C.; Sales, M. Goreti F.

    2014-01-01

    This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice...

  10. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  11. Auto-FACE: an NMR based binding site mapping program for fast chemical exchange protein-ligand systems.

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    Janarthanan Krishnamoorthy

    Full Text Available BACKGROUND: Nuclear Magnetic Resonance (NMR spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQCexperiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. METHODOLOGY/PRINCIPAL FINDINGS: We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues.Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBcl(XL and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3.0 x 10(4 calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12.8 x 10(4. Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker

  12. Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

    Directory of Open Access Journals (Sweden)

    Irina N Marinova

    Full Text Available The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.

  13. Insight into herbicide resistance of W574L mutant Arabidopsis thaliana acetohydroxyacid synthase:molecular dynamics simulations and binding free energy calculations

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Acetohydroxyacid synthase(AHAS) is the target enzyme of several classes of herbicides,such as sulfonylureas and imidazolinones.Now many mutant AHASs with herbicide resistance have emerged along with extensive use of herbicides,therefore it is imperative to understand the detailed interaction mechanism and resistance mechanism so as to develop new potent inhibitors for wild-type or resistant AHAS.With the aid of available crystal structures of the Arabidopsis thaliana(At) AHAS-inhibitor complex,molecular dynamics(MD) simulations were used to investigate the interaction and resistance mechanism directly and dynamically at the atomic level.Nanosecond-level MD simulations were performed on six systems consisting of wild-type or W574L mutant AtAHAS in the complex with three sulfonylurea inhibitors,separately,and binding free energy was calculated for each system using the MM-GBSA method.Comprehensive analyses from structural and energetic aspects confirmed the importance of residue W574,and also indicated that W574L mutation might alert the structural charactersistic of the substrate access channel and decrease the binding affinity of inhibitors,which cooperatively weaken the effective channel-blocked effect and finally result in weaker inhibitory effect of inhibitor and corresponding herbicide resistance of W574L mutant.To our knowledge,it is the first report about MD simulations study on the AHAS-related system,which will pave the way to study the interactions between herbicides and wild-type or mutant AHAS dynamically,and decipher the resistance mechanism at the atomic level for better designing new potent anti-resistance herbicides.

  14. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen (GSKPA)

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  15. Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Okada

    Full Text Available In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4 is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

  16. Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

    OpenAIRE

    Grigoryan, Hasmik; Li, Bin; Anderson, Erica K.; Xue, Weihua; Nachon, Florian; Lockridge, Oksana; Schopfer, Lawrence M.

    2009-01-01

    Organophosphorus esters (OP) are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-b...

  17. Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice.

    Science.gov (United States)

    Watanabe, Shigeru; Yamamori, Saori; Otsuka, Shintaro; Saito, Masanori; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

    2015-09-01

    Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of

  18. Adaptation of human immunodeficiency virus type 1 to cells expressing a binding-deficient CD4 mutant (lysine 46 to aspartic acid).

    Science.gov (United States)

    Choe, H R; Sodroski, J

    1995-01-01

    Human immunodeficiency virus (HIV-1) was adapted to replicate efficiently in cells expressing an altered form of the CD4 viral receptor. The mutant CD4 (46 K/D) contained a single amino acid change (lysine 46 to aspartic acid) in the CDR2 loop of domain 1, which results in a 15-fold reduction in affinity for the viral gp120 glycoprotein. The ability of the adapted virus to replicate in CD4 46 K/D-expressing cells was independently enhanced by single amino acid changes in the V2 variable loop, the V3 variable loop, and the fourth conserved (C4) region of the gp120 glycoprotein. Combinations of these amino acids in the same envelope glycoprotein resulted in additive enhancement of virus replication in cells expressing the CD4 46 K/D molecule. In cells expressing the wild-type CD4 glycoproteins, the same V2 and V3 residue changes also increased the efficiency of replication of a virus exhibiting decreased receptor-binding ability due to an amino acid change (aspartic acid 368 to glutamic acid) in the gp120 glycoprotein. In neither instance did the adaptive changes restore the binding ability of the monomeric gp120 glycoprotein or the oligomeric envelope glycoprotein complex for the mutant or wild-type CD4 glycoproteins, respectively. Thus, particular conformations of the gp120 V2 and V3 variable loops and of the C4 region allow postreceptor binding events in the membrane fusion process to occur in the context of less than optimal receptor binding. These results suggest that the fusion-related functions of the V2, V3, and C4 regions of gp120 are modulated by CD4 binding. PMID:7707502

  19. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    Science.gov (United States)

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2014-10-24

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

  20. The role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    CERN Document Server

    Karapetyan, Sargis

    2015-01-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ul...