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Sample records for active site ligand

  1. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  2. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  3. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  4. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  5. Modulating protein activity using tethered ligands with mutually exclusive binding sites

    PubMed Central

    Schena, Alberto; Griss, Rudolf; Johnsson, Kai

    2015-01-01

    The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors. PMID:26198003

  6. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO

    NASA Astrophysics Data System (ADS)

    Alfonso, Dominic R.; Kauffman, Douglas; Matranga, Christopher

    2016-05-01

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25- nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts.

  7. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO.

    PubMed

    Alfonso, Dominic R; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25 (-) nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts. PMID:27179498

  8. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha

  9. Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

    PubMed

    Krishna Das, Bhaba; Kumar, Amit; Maindola, Priyank; Mahanty, Srikrishna; Jain, S K; Reddy, Mallireddy K; Arockiasamy, Arulandu

    2016-05-13

    Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. PMID:27067046

  10. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site

    PubMed Central

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J.; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I.; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A.; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-01-01

    Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. PMID:25672491

  11. Ligand Displacement Reaction Paths in a Diiron Hydrogenase Active Site Model Complex.

    PubMed

    Blank, Jan H; Moncho, Salvador; Lunsford, Allen M; Brothers, Edward N; Darensbourg, Marcetta Y; Bengali, Ashfaq A

    2016-08-26

    The mechanism and energetics of CO, 1-hexene, and 1-hexyne substitution from the complexes (SBenz)2 [Fe2 (CO)6 ] (SBenz=SCH2 Ph) (1-CO), (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexene)] (1-(η(2) -1-hexene)), and (SBenz)2 [Fe2 (CO)5 (η(2) -1-hexyne)] (1-(η(2) -1-hexyne)) were studied by using time-resolved infrared spectroscopy. Exchange of both CO and 1-hexyne by P(OEt)3 and pyridine, respectively, proceeds by a bimolecular mechanism. As similar activation enthalpies are obtained for both reactions, the rate-determining step in both cases is assumed to be the rotation of the Fe(CO)2 L (L=CO or 1-hexyne) unit to accommodate the incoming ligand. The kinetic profile for the displacement of 1-hexene is quite different than that for the alkyne and, in this case, both reaction channels, that is, dissociative (SN 1) and associative (SN 2), were found to be competitive. Because DFT calculations predict similar binding enthalpies of alkene and alkyne to the iron center, the results indicate that the bimolecular pathway in the case of the alkyne is lower in free energy than that of the alkene. In complexes of this type, subtle changes in the departing ligand characteristics and the nature of the mercapto bridge can influence the exchange mechanism, such that more than one reaction pathway is available for ligand substitution. The difference between this and the analogous study of (μ-pdt)[Fe(CO)3 ]2 (pdt=S(CH2 )3 S) underscores the unique characteristics of a three-atom S-S linker in the active site of diiron hydrogenases. PMID:27482938

  12. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

    PubMed

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  13. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  14. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  15. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

    PubMed

    Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Kelly S; Singh, Sandip; Bowman, Lesley Ah; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  16. Crystal Structure, Exogenous Ligand Binding and Redox Properties of an Engineered Diiron Active Site in a Bacterial Hemerythrin

    PubMed Central

    Okamoto, Yasunori; Onoda, Akira; Sugimoto, Hiroshi; Takano, Yu; Hirota, Shun; Kurtz, Donald M.; Shiro, Yoshitsugu; Hayashi, Takashi

    2013-01-01

    A non-heme diiron active site in a 13-kDa hemerythrin-like domain of the bacterial chemotaxis protein, DcrH-Hr, contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands, azide and phenol, to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical non-heme diiron-carboxylate protein active site. PMID:24187962

  17. A Cu4S model for the nitrous oxide reductase active sites supported only by nitrogen ligands.

    PubMed

    Johnson, Brittany J; Antholine, William E; Lindeman, Sergey V; Mankad, Neal P

    2015-07-28

    To model the (His)7Cu4Sn (n = 1 or 2) active sites of nitrous oxide reductase, the first Cu4(μ4-S) cluster supported only by nitrogen donors has been prepared using amidinate supporting ligands. Structural, magnetic, spectroscopic, and computational characterization is reported. Electrochemical data indicates that the 2-hole model complex can be reduced reversibly to the 1-hole state and irreversibly to the fully reduced state. PMID:26111160

  18. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

  19. Flavonol Activation Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase Domain of IRE1

    SciTech Connect

    Wiseman, R. Luke; Zhang, Yuhong; Lee, Kenneth P.K.; Harding, Heather P.; Haynes, Cole M.; Price, Joshua; Sicheri, Frank; Ron, David

    2010-08-18

    Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.

  20. A semisynthetic strategy leads to alteration of the backbone amidate ligand in the NiSOD active site

    SciTech Connect

    Campeciño, Julius O.; Dudycz, Lech W.; Tumelty, David; Berg, Volker; Cabelli, Diane E.; Maroney, Michael J.

    2015-07-01

    Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ~1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ~11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. 14N-hyperfine is observed on gzligand in the Ni(III) complex. As a result, the altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models.

  1. A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site.

    PubMed

    Campeciño, Julius O; Dudycz, Lech W; Tumelty, David; Berg, Volker; Cabelli, Diane E; Maroney, Michael J

    2015-07-22

    Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ∼1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. (14)N-hyperfine is observed on gz, confirming the addition of the apical histidine ligand in the Ni(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models. PMID:26135142

  2. A semisynthetic strategy leads to alteration of the backbone amidate ligand in the NiSOD active site

    DOE PAGESBeta

    Campeciño, Julius O.; Dudycz, Lech W.; Tumelty, David; Berg, Volker; Cabelli, Diane E.; Maroney, Michael J.

    2015-07-01

    Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategymore » that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ~1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ~11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. 14N-hyperfine is observed on gz« less

  3. Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase

    SciTech Connect

    Ronning, Donald R; Iacopelli, Natalie M; Mishra, Vidhi

    2012-03-15

    The bacterial enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a central role in three essential metabolic pathways in bacteria: methionine salvage, purine salvage, and polyamine biosynthesis. Recently, its role in the pathway that leads to the production of autoinducer II, an important component in quorum-sensing, has garnered much interest. Because of this variety of roles, MTAN is an attractive target for developing new classes of inhibitors that influence bacterial virulence and biofilm formation. To gain insight toward the development of new classes of MTAN inhibitors, the interactions between the Helicobacter pylori-encoded MTAN and its substrates and substrate analogs were probed using X-ray crystallography. The structures of MTAN, an MTAN-Formycin A complex, and an adenine bound form were solved by molecular replacement and refined to 1.7, 1.8, and 1.6 Å, respectively. The ribose-binding site in the MTAN and MTAN-adenine cocrystal structures contain a tris[hydroxymethyl]aminomethane molecule that stabilizes the closed form of the enzyme and displaces a nucleophilic water molecule necessary for catalysis. This research gives insight to the interactions between MTAN and bound ligands that promote closing of the enzyme active site and highlights the potential for designing new classes of MTAN inhibitors using a link/grow or ligand assembly development strategy based on the described H. pylori MTAN crystal structures.

  4. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed. PMID:24484052

  5. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    NASA Astrophysics Data System (ADS)

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  6. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    PubMed Central

    Holby, Edward F.; Taylor, Christopher D.

    2015-01-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date. PMID:25788358

  7. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: role of *OH ligands.

    PubMed

    Holby, Edward F; Taylor, Christopher D

    2015-01-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date. PMID:25788358

  8. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    DOE PAGESBeta

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

  9. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    SciTech Connect

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  10. The intriguing enhancement of chloroperoxidase mediated one-electron oxidations by azide, a known active-site ligand

    SciTech Connect

    Andrew, Daniel; Hager, Lowell; Manoj, Kelath Murali

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Azide is a well known heme-enzyme active site ligand and inhibitor. Black-Right-Pointing-Pointer Herein, azide is reported to enhance a set of heme-enzyme mediated reactions. Black-Right-Pointing-Pointer This effect is disconnected from native enzyme-azide binding. Black-Right-Pointing-Pointer Azide could enhance heme-enzyme reactions via a newly proposed mechanism. Black-Right-Pointing-Pointer Azide contained in reagents could impact reaction outcomes in redox biochemistry. -- Abstract: Azide is a well-known inhibitor of heme-enzymes. Herein, we report the counter-intuitive observation that at some concentration regimes, incorporation of azide in the reaction medium enhances chloroperoxidase (CPO, a heme-enzyme) mediated one-electron abstractions from several substrates. A diffusible azidyl radical based mechanism is proposed for explaining the phenomenon. Further, it is projected that the finding could have significant impact on routine in situ or in vitro biochemistry studies involving heme-enzyme systems and azide.

  11. A ligand field chemistry of oxygen generation by the oxygen-evolving complex and synthetic active sites

    PubMed Central

    Betley, Theodore A; Surendranath, Yogesh; Childress, Montana V; Alliger, Glen E; Fu, Ross; Cummins, Christopher C; Nocera, Daniel G

    2007-01-01

    Oxygen–oxygen bond formation and O2 generation occur from the S4 state of the oxygen-evolving complex (OEC). Several mechanistic possibilities have been proposed for water oxidation, depending on the formal oxidation state of the Mn atoms. All fall under two general classifications: the AB mechanism in which nucleophilic oxygen (base, B) attacks electrophilic oxygen (acid, A) of the Mn4Ca cluster or the RC mechanism in which radical-like oxygen species couple within OEC. The critical intermediate in either mechanism involves a metal oxo, though the nature of this oxo for AB and RC mechanisms is disparate. In the case of the AB mechanism, assembly of an even-electron count, high-valent metal-oxo proximate to a hydroxide is needed whereas, in an RC mechanism, two odd-electron count, high-valent metal oxos are required. Thus the two mechanisms give rise to very different design criteria for functional models of the OEC active site. This discussion presents the electron counts and ligand geometries that support metal oxos for AB and RC O–O bond-forming reactions. The construction of architectures that bring two oxygen functionalities together under the purview of the AB and RC scenarios are described. PMID:17971328

  12. Simultaneous binding of coenzyme and two ligand molecules into the active site of fungal trihydroxynaphthalene reductase.

    PubMed

    Stojan, Jure; Brunskole, Mojca; Rizner, Tea Lanisnik

    2009-03-16

    We present here a kinetic characterization of the oxidation of the artificial substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one in the presence of NADP(+) by trihydroxynaphthalene reductase from the filamentous fungus Curvularia lunata. Although the experimental data were gathered by conventional equipment and were only available for the reaction in one direction, the analysis confirms the bi-bi reaction mechanism and yields estimates of kinetic parameters of the intermediates. It is based on an independent estimation of coenzyme binding constants and on a sequential analysis of three portions of the progress curves, from the beginning of the reaction until equilibrium is reached. First, the plateaus are used to determine the overall equilibrium constant of the non-catalyzed reaction. Then, the dissociation constants of the oxidized and reduced cofactor are estimated by titration. Subsequently, the initial parts of the progress curves are analyzed using the rate equation that is derived under combined assumptions of equilibrium and steady state. The macroscopic relations obtained are then fixed in the final progress curve analysis where the information for only two remaining rate constants is extracted from their curved portions by fitting numerically solved model-specific differential equations to the data. At pH 8, the overall equilibrium largely favours the oxidized substrate and reduced cofactor, and the activity of the holoenzyme is inhibited by high substrate concentrations. Substrate inhibition can be discriminated from true cooperativity through the effects of apigenin, a flavonoid inhibitor that is structurally similar, but larger, than the substrate used in the study. PMID:19071099

  13. Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand.

    PubMed

    Thielges, Megan C; Axup, Jun Y; Wong, Daryl; Lee, Hyun Soo; Chung, Jean K; Schultz, Peter G; Fayer, Michael D

    2011-09-29

    Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein. PMID:21823631

  14. Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site Ligand

    PubMed Central

    Thielges, Megan C.; Axup, Jun Y.; Wong, Daryl; Lee, Hyun Soo; Chung, Jean K.; Schultz, Peter G.; Fayer, Michael D.

    2012-01-01

    Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ~1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein. PMID:21823631

  15. Predicting target-ligand interactions using protein ligand-binding site and ligand substructures

    PubMed Central

    2015-01-01

    Background Cell proliferation, differentiation, Gene expression, metabolism, immunization and signal transduction require the participation of ligands and targets. It is a great challenge to identify rules governing molecular recognition between chemical topological substructures of ligands and the binding sites of the targets. Methods We suppose that the ligand-target interactions are determined by ligand substructures as well as the physical-chemical properties of the binding sites. Therefore, we propose a fragment interaction model (FIM) to describe the interactions between ligands and targets, with the purpose of facilitating the chemical interpretation of ligand-target binding. First we extract target-ligand complexes from sc-PDB database, based on which, we get the target binding sites and the ligands. Then we represent each binding site as a fragment vector based on a target fragment dictionary that is composed of 199 clusters (denoted as fragements in this work) obtained by clustering 4200 trimers according to their physical-chemical properties. And then, we represent each ligand as a substructure vector based on a dictionary containing 747 substructures. Finally, we build the FIM by generating the interaction matrix M (representing the fragment interaction network), and the FIM can later be used for predicting unknown ligand-target interactions as well as providing the binding details of the interactions. Results The five-fold cross validation results show that the proposed model can get higher AUC score (92%) than three prevalence algorithms CS-PD (80%), BLM-NII (85%) and RF (85%), demonstrating the remarkable predictive ability of FIM. We also show that the ligand binding sites (local information) overweight the sequence similarities (global information) in ligand-target binding, and introducing too much global information would be harmful to the predictive ability. Moreover, The derived fragment interaction network can provide the chemical insights on

  16. Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate.

    PubMed

    Lummis, Sarah C R; Fletcher, Elizabeth J; Green, Tim

    2002-03-01

    Activation of N-methyl-d-aspartate (NMDA) receptors requires the binding of both glutamate and glycine to independent sites on the receptor. These ligands bind to NR2 and NR1 subunits respectively. Ligand binding residues are located in two non-contiguous domains, S1 and S2, which have been implicated in glutamate binding in other ionotropic glutamate receptor subunits. To further define the amino acids through which glutamate activates the receptor, we generated single-site mutations to the NR2A subunit, and expressed them with wild type NR1 in HEK 293 cells. Using calcium imaging and whole cell patch clamp we determined glutamate and glycine potencies. Of the eight residues mutated we identified five (E413, K484, A508, G685 and G688), whose mutation leads to a large reduction (from 4- to 1000-fold) in glutamate potency, consistent with a role for these residues in receptor activation by glutamate. The potency of glycine was largely unchanged by these mutations. Thus our results extend the knowledge base of residues involved in NMDA receptor function and identifies a new site in S1, in the region of A508, that has a role in receptor activation by glutamate. PMID:11955515

  17. Supramolecular Chemistry And Self-assembly Special Feature: Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

    NASA Astrophysics Data System (ADS)

    Hodneland, Christian D.; Lee, Young-Sam; Min, Dal-Hee; Mrksich, Milan

    2002-04-01

    This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

  18. Sterically hindered carboxylate ligands support water-bridged dimetallic centers that model features of metallohydrolase active sites.

    PubMed

    Lee, Dongwhan; Hung, Pei-Lin; Spingler, Bernhard; Lippard, Stephen J

    2002-02-11

    The synthesis and characterization of carboxylate-bridged dimetallic complexes are described. By using m-terphenyl-derived carboxylate ligands, a series of dicobalt(II), dicobalt(III), dinickel(II), and dizinc(II) complexes were synthesized. The compounds are [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (1), [Co(2)(mu-OH(2))(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (2a-c), [Co(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (3), [Ni(2)(mu-O(2)CAr(Tol))(4)L(2)] (4), [Ni(2)(mu-HO...H)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (5), and [Zn(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (6), where Ar(Tol)CO(2)H = 2,6-di(p-tolyl)benzoic acid and L = pyridine, THF, or N,N-dibenzylethylenediamine. Structural analysis of these complexes revealed that additional bridging ligands can be readily accommodated within the [M(2)(mu-O(2)CAr(Tol))(2)](2+) core, allowing a wide distribution of M...M distances from 2.5745(6) to 4.0169(9) A. Unprecedented bridging units [M(2)(mu-OH(2))(2)(mu-O(2)CR)(2)](n+) and [M(2)(mu-HO...H)(2)(mu-O(2)CR)(2)](n+) were identified in 2a-c and 5, respectively, in which strong hydrogen bonding accommodates shifts of protons from bridging water molecules toward the dangling oxygen atoms of terminal monodentate carboxylate groups. Such a proton shift along the O...H...O coordinate attenuates the donor ability of the anionic carboxylate ligand, which can translate into increased Lewis acidity at the metal centers. Such double activation of bridging water molecules by a Lewis acidic metal center and a metal-bound general base may facilitate the reactivity of metallohydrolases such as methionine aminopeptidase (MAP). PMID:11825079

  19. Calculation of Vibrational Shifts of Nitrile Probes in the Active Site of Ketosteroid Isomerase upon Ligand Binding

    PubMed Central

    Layfield, Joshua P.

    2012-01-01

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analog equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active site water molecule that is directly hydrogen bonded to the nitrile probe, resulting in a more linear CNH angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis. PMID:23210919

  20. Fluorescence properties and sequestration of peripheral anionic site specific ligands in bile acid hosts: Effect on acetylcholinesterase inhibition activity.

    PubMed

    Islam, Mullah Muhaiminul; Aguan, Kripamoy; Mitra, Sivaprasad

    2016-05-01

    The increase in fluorescence intensity of model acetyl cholinesterase (AChE) inhibitors like propidium iodide (PI) and ethidium bromide (EB) is due to sequestration of the probes in primary micellar aggregates of bile acid (BA) host medium with moderate binding affinity of ca. 10(2)-10(3)M(-1). Multiple regression analysis of solvent dependent fluorescence behavior of PI indicates the decrease in total nonradiative decay rate due to partial shielding of the probe from hydrogen bond donation ability of the aqueous medium in bile acid bound fraction. Both PI and EB affects AChE activity through mixed inhibition and consistent with one site binding model; however, PI (IC50=20±1μM) shows greater inhibition in comparison with EB (IC50=40±3μM) possibly due to stronger interaction with enzyme active site. The potency of AChE inhibition for both the compounds is drastically reduced in the presence of bile acid due to the formation of BA-inhibitor complex and subsequent reduction of active inhibitor fraction in the medium. Although the inhibition mechanism still remains the same, the course of catalytic reaction critically depends on equilibrium binding among several species present in the solution; particularly at low inhibitor concentration. All the kinetic parameters for enzyme inhibition reaction are nicely correlated with the association constant for BA-inhibitor complex formation. PMID:26974580

  1. Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor crucial for ligand-induced internalization.

    PubMed

    Hull, J J; Lee, J M; Matsumoto, S

    2011-12-01

    Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Using fluorescent Bombyx mori PBANR (BmPBANR) chimeras to study PBANR regulation, we previously showed that BmPBANR undergoes rapid ligand-induced internalization, that the endocytotic motif resides between residues 358-367 of the BmPBANR C terminus, and that the internalization pathway is clathrin-dependent. Here, we sought to expand our understanding of the molecular mechanisms underlying BmPBANR function and regulation by transiently expressing a series of fluorescent BmPBANR chimeric constructs in cultured Spodoptera frugiperda (Sf9) cells and assaying for internalization of a fluorescently labelled ligand. Pharmacological inhibition of phospholipase C significantly reduced internalization, suggesting that BmPBANR regulation proceeds via a conventional G-protein-dependent pathway. This was further supported by impaired internalization following site-directed mutagenesis of R263 and R264, two basic residues at the transmembrane 6 intracellular junction that are thought to stabilize G-protein coupling via electrostatic interactions. Ala substitution of S333 and S366, two consensus protein kinase C sites in the C terminus, likewise impaired internalization, as did RNA interference-mediated knockdown of Sf9 protein kinase C. N-terminal truncations of BmPBANR indicate that the first 27 residues are not necessary for cell surface trafficking or receptor functionality. PMID:21955122

  2. Active-Site Models for the Nickel-Iron Hydrogenases: Effects of Ligands on Reactivity and Catalytic Properties

    PubMed Central

    Carroll, Maria E.; Barton, Bryan E.; Gray, Danielle L.; Mack, Amanda E.; Rauchfuss, Thomas B.

    2011-01-01

    Described are new derivatives of the type [HNiFe(SR)2(diphosphine)(CO)3]+, which feature a Ni(diphosphine) group linked to a Fe(CO)3 group via two bridging thiolate ligands. Previous work had described [HNiFe(pdt)(dppe)(CO)3]+ ([1H]+) and its activity as a catalyst for the reduction of protons. Work described in this paper focused on the effects of the diphosphine attached to nickel as well as the dithiolate bridge, 1,3-propanedithiolate (pdt) vs 1,2-ethanedithiolate (edt). A new synthetic route to these Ni-Fe dithiolates is described, involving reaction of Ni(SR)2(diphosphine) with FeI2(CO)4 followed by in situ reduction with cobaltocene. Evidence is presented that this route proceeds via metastable μ-iodo derivatives. Attempted isolation of such species led to the crystallization of NiFe(Me2pdt)(dppe)I2, which features tetrahedral Fe(II) and square planar Ni(II) centers (Me2pdt = 2,2-dimethylpropanedithiol). The new tricarbonyls prepared in this work are NiFe(pdt)(dcpe)(CO)3 (2, dcpe = 1,2-bis(dicyclohexylphosphino)ethane), NiFe(edt)(dppe)(CO)3 (3), and NiFe(edt)(dcpe)(CO)3 (4). Attempted preparation of a phenylthiolate-bridged complex via the FeI2(CO)4 + Ni(SPh)2(dppe) route gave the tetrametallic species [(CO)2Fe(SPh)2Ni(CO)]2(μ-dppe)2. Crystallographic analysis of the edt-dcpe compund [2H]BF4 and the edt-dppe compound [3H]BF4 verified their close resemblance. Each features pseudo-octahedral Fe and square pyramidal Ni centers. Starting from [4H]BF4 we prepared the PPh3 derivative [HNiFe(edt)(dppe)(PPh3)(CO)2]BF4 ([5H]BF4), which was obtained as a ~2:1 mixture of unsymmetrical and symmetrical isomers. Acid-base measurements indicate that changing from Ni(dppe) to Ni(dcpe) decreases the acidity of the cationic hydride complexes by 2.5 pKaMeCN units, from ~11 to ~13.5 (previous work showed that substitution at Fe leads to more dramatic effects). The redox potentials are more strongly affected by the change from dppe to dcpe, for example the [2]0/+ couple occurs

  3. Sulfur K-edge XAS and DFT Studies on NiII Complexes with Oxidized Thiolate Ligands: Implications for the Roles of Oxidized Thiolates in the Active Sites of Fe and Co Nitrile Hydratase

    PubMed Central

    Dey, Abhishek; Jeffrey, Stephen P.; Darensbourg, Marcetta; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.

    2008-01-01

    S K-edge XAS data on a series of NiII complexes with thiolate (RS−) and oxidized thiolate (RSO2−) ligands are used to quantify Ni-S bond covalency and its change upon ligand oxidation. Analyses of these results using geometry optimized DFT calculations suggest that the Ni-S σ bonds do not weaken on ligand oxidation. Molecular orbital analysis indicates that these oxidized thiolate ligands use filled high lying S-O π* orbitals for strong σ donation. However, the RSO2− ligands are poor π donors as the orbital required for π interaction is is used in the S-O σ bond formation. The oxidation of the thiolate reduces the repulsion between electrons in the filled Ni t2 orbital and the thiolate out of plane π-donor orbital leading to shorter Ni-S bond length relative to a thiolate donor. The insights obtained from these results are relevant to the active sites of Fe and Co type nitrile hydratases (Nhase) that also have oxidized thiolate ligands. DFT calculations on models of the active site indicate that while the oxidation of these thiolates has a major effect in the axial ligand binding affinity of the Fe type Nhase (where there is both σ and π donation from the S ligands), it has only a limited effect on the sixth ligand binding affinity of the Co type Nhases (where there is only σ donation). These oxidized residues may also play a role in substrate binding and proton shuttling at the active site. PMID:17500514

  4. FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.

    PubMed

    Arcos-Hernández, César; Romero, Francisco; Sánchez-Guevara, Yoloxochitl; Beltrán, Carmen; Nishigaki, Takuya

    2016-02-01

    Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l(-1) eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca(2+) concentrations similar to those triggered by 10 nmol l(-1) speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor. PMID:26889001

  5. Blockade by sigma site ligands of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones.

    PubMed Central

    Church, J.; Fletcher, E. J.

    1995-01-01

    1. The effects of a series of structurally-dissimilar sigma site ligands were examined on high voltage-activated Ca2+ channel activity in two preparations of cultured hippocampal pyramidal neurones. 2. In mouse hippocampal neurones under whole-cell voltage-clamp, voltage-activated Ca2+ channel currents carried by barium ions (IBa) were reduced with the rank order (IC50 values in microM): 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]- 2-(1-pyrrolidinyl)cyclohexylamine (7.8) > rimcazole (13) > haloperidol (16) > ifenprodil (18) > opipramol (32) > carbetapentane (40) = 1-benzylspiro[1,2,3,4-tetrahydronaphthalene-1,4-piperidine] (42) > caramiphen (47) > dextromethorphan (73). At the highest concentrations tested, the compounds almost abolished IBa in the absence of any other pharmacological agent. 3. The current-voltage characteristics of the whole-cell IBa were unaffected by the test compounds. The drug-induced block was rapid in onset and offset, with the exceptions of carbetapentane and caramiphen where full block was achieved only after two to three voltage-activated currents and was associated with an apparent increase in the rate of inactivation of IBa. 4. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, rises in intracellular free Ca2+ concentration evoked by transient exposure to 50 mM K(+)-containing medium, either in the absence or in the presence of 10 microM nifedipine (to block L-type high voltage-activated Ca2+ channels), were also reversibly attenuated by the sigma ligands. The rank order potencies for the compounds in these experimental paradigms were similar to that observed for blockade of IBa in the electrophysiological studies. 5. These results indicate that, at micromolar concentrations, the compounds tested block multiple subtypes of high voltage-activated Ca2+ channels. These actions, which do not appear to be mediated by high-affinity sigma binding sites, may play a role in some of the functional effects

  6. Follitropin receptors contain cryptic ligand binding sites.

    PubMed

    Lin, Win; Bernard, Michael P; Cao, Donghui; Myers, Rebecca V; Kerrigan, John E; Moyle, William R

    2007-01-01

    Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

  7. The Binding Mode Prediction and Similar Ligand Potency in the Active Site of Vitamin D Receptor with QM/MM Interaction, MESP, and MD Simulation.

    PubMed

    Selvaraman, Nagamani; Selvam, Saravana Kumar; Muthusamy, Karthikeyan

    2016-08-01

    Non-secosteroidal ligands are well-known vitamin D receptor (VDR) agonists. In this study, we described a combined QM/MM to define the protein-ligand interaction energy a strong positive correlation in both QM-MM interaction energy and binding free energy against the biological activity. The molecular dynamics simulation study was performed, and specific interactions were extensively studied. The molecular docking results and surface analysis shed light on steric and electrostatic complementarities of these non-secosteroidal ligands to VDR. Finally, the drug likeness properties were also calculated and found within the acceptable range. The results show that bulky group substitutions in side chain decrease the VDR activity, whereas a small substitution increased it. Functional analyses of H393A and H301A mutations substantiate their roles in the VDR agonistic and antagonistic activities. Apart from the His393 and His301, two other amino acids in the hinge region viz. Ser233 and Arg270 acted as an electron donor/acceptor specific to the agonist in the distinct ligand potency. The results from this study disclose the binding mechanism of VDR agonists and structural modifications required to improve the selectivity. PMID:26945790

  8. Postsynthetic Metal and Ligand Exchange in MFU-4l: A Screening Approach toward Functional Metal-Organic Frameworks Comprising Single-Site Active Centers.

    PubMed

    Denysenko, Dmytro; Jelic, Jelena; Reuter, Karsten; Volkmer, Dirk

    2015-05-26

    The isomorphous partial substitution of Zn(2+) ions in the secondary building unit (SBU) of MFU-4l leads to frameworks with the general formula [M(x)Zn(5-x)Cl4(BTDD)3], in which x≈2, M = Mn(II), Fe(II), Co(II), Ni(II), or Cu(II), and BTDD = bis(1,2,3-triazolato-[4,5-b],[4',5'-i])dibenzo-[1,4]-dioxin. Subsequent exchange of chloride ligands by nitrite, nitrate, triflate, azide, isocyanate, formate, acetate, or fluoride leads to a variety of MFU-4l derivatives, which have been characterized by using XRPD, EDX, IR, UV/Vis-NIR, TGA, and gas sorption measurements. Several MFU-4l derivatives show high catalytic activity in a liquid-phase oxidation of ethylbenzene to acetophenone with air under mild conditions, among which Co- and Cu derivatives with chloride side-ligands are the most active catalysts. Upon thermal treatment, several side-ligands can be transformed selectively into reactive intermediates without destroying the framework. Thus, at 300 °C, Co(II)-azide units in the SBU of Co-MFU-4l are converted into Co(II)-isocyanate under continuous CO gas flow, involving the formation of a nitrene intermediate. The reaction of Cu(II)-fluoride units with H2 at 240 °C leads to Cu(I) and proceeds through the heterolytic cleavage of the H2 molecule. PMID:25882594

  9. Effect of the Active Site D25N Mutation on the Structure, Stability and Ligand Binding of the Mature HIV-1 Protease

    SciTech Connect

    Sayer, Jane M.; Liu, Fengling; Ishima, Rieko; Weber, Irene T.; Louis, John M.

    2008-09-03

    All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR{sub D25N}) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 {+-} 0.09 {mu}m) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR{sub D25N} in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 C, respectively. Only minimal differences are observed in high resolution crystal structures of PR{sub D25N} complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH{sub 2} (RPB), as compared with PR{center_dot}DRV and PR{center_dot}RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T{sub m} is smaller for PR{sub D25N} (6.2 C) than for PR (8.7 C). The T{sub m} of PR{sub D25N}{center_dot}DRV increases by only 3 C relative to free PR{sub D25N}, as compared with a 22 C increase for PR{center_dot}DRV, and the mutation increases the ligand dissociation constant of PR{sub D25N}{center_dot}DRV by a factor of {approx}10{sup 6} relative to PR{center_dot}DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

  10. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    PubMed

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function. PMID:26064949

  11. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites

    PubMed Central

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where “nonspecific” interactions contribute to biological function. PMID:26064949

  12. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  13. Regulation of the extracellular ligand binding activity of integrins.

    PubMed

    Fernandez, C; Clark, K; Burrows, L; Schofield, N R; Humphries, M J

    1998-07-01

    Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed. PMID:9637803

  14. Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Moraxella catarrhalis reveal a water channel connecting to the active site for the second step of catalysis.

    PubMed

    Dhindwal, Sonali; Priyadarshini, Priyanka; Patil, Dipak N; Tapas, Satya; Kumar, Pramod; Tomar, Shailly; Kumar, Pravindra

    2015-02-01

    KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC from Moraxella catarrhalis (Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for the Mc-KdsC enzyme. PMID:25664734

  15. Structures of the Wild-Type And Activated Catalytic Domains of Brachydanio Rerio Polo-Like Kinase 1 (Plk1): Changes in the Active-Site Conformation And Interactions With Ligands

    SciTech Connect

    Elling, R.A.; Fucini, R.V.; Romanowski, M.J.

    2009-05-18

    Polo-like kinase 1 (Plk1) is a member of a family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. A zebrafish homolog of the human Plk1 (hPlk1) kinase domain (KD) was identified that can be expressed in large quantities in bacteria and crystallizes readily, whether in a wild-type form or as a variant containing the activating Thr196-->Asp substitution, in one space group and under similar conditions both in the absence and presence of active-site compounds. This construct was validated by testing a panel of hPlk1 inhibitors against human and zebrafish proteins and it was shown that the selected small molecules inhibited the homologs with a high degree of correlation. Crystal structures of ligand-free wild-type and activated zebrafish Plk1 (zPlk1) KDs revealed the organization of the secondary structural elements around the active site and demonstrated that the activation segment was disordered in the activated form of the domain but possessed a well defined secondary structure in the wild-type enzyme. The cocrystal structure of wild-type zPlk1 KD with ADP documented the hydrolysis of ATP and revealed the phosphorylation site. The cocrystal structure of the activated KD with wortmannin, a covalent inhibitor of Plk1 and PI3 kinases, showed the binding mode of the small molecule to the enzyme and may facilitate the design of more potent Plk1 inhibitors. The work presented in this study establishes the zPlk1 KD as a useful tool for rapid low- and high-throughput structure-based screening and drug discovery of compounds specific for this mitotic target.

  16. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    PubMed

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS. PMID:18820259

  17. Cloud Computing for Protein-Ligand Binding Site Comparison

    PubMed Central

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

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

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  19. Crystal structure of the dithiol oxidase DsbA enzyme from proteus mirabilis bound non-covalently to an active site peptide ligand.

    PubMed

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A; Fairlie, David P; Martin, Jennifer L

    2014-07-11

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  20. Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

    PubMed Central

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

    2014-01-01

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  1. Design, synthesis and evaluation of rivastigmine and curcumin hybrids as site-activated multitarget-directed ligands for Alzheimer's disease therapy.

    PubMed

    Li, Yujie; Peng, Peng; Tang, Li; Hu, Yunzhen; Hu, Yongzhou; Sheng, Rong

    2014-09-01

    A series of novel 2-methoxy-phenyl dimethyl-carbamate derivatives were designed, synthesized and evaluated as site-activated MTDLs based on rivastigmine and curcumin. Most of them exhibited good to excellent AChE and BuChE inhibitory activities with sub-micromolar IC50 values. Among all the compounds, 6a demonstrated the most potent AChE inhibition with IC50 value of 0.097μM, which is about 20-fold than that of rivastigmine. In addition, the three selected compounds 5a, 6a and 6e demonstrated inhibitory activity against Aβ self-aggregation similar to cucurmin in TEM assay, which is obviously different from the weak activity of rivastigmine. Moreover, the hydrolysate of 6a (compound 7) also showed potent ABTS(+) scavenging and moderate copper ion chelating activity in vitro. PMID:25082512

  2. Role of Enzyme Flexibility in Ligand Access and Egress to Active Site: Bias-Exchange Metadynamics Study of 1,3,7-Trimethyluric Acid in Cytochrome P450 3A4.

    PubMed

    Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Laio, Alessandro; Otyepka, Michal

    2016-04-12

    Although the majority of enzymes have buried active sites, very little is known about the energetics and mechanisms associated with substrate and product channeling in and out. Gaining direct information about these processes is a challenging task both for experimental and theoretical techniques. Here, we present a methodology that enables following of a ligand during its passage to the active site of cytochrome P450 (CYP) 3A4 and mapping of the free energy associated with this process. The technique is based on a combination of a bioinformatics tool for identifying access channels and bias-exchange metadynamics and provides converged free energies in good agreement with experimental data. In addition, it identifies the energetically preferred escape routes, limiting steps, and amino acids residues lining the channel. The approach was applied to mapping of a complex channel network in a complex environment, i.e., CYP3A4 attached to a lipid bilayer mimicking an endoplasmic reticulum membrane. The results provided direct information about the energetics and conformational changes associated with the ligand channeling. The methodology can easily be adapted to study channeling through other flexible biomacromolecular channels. PMID:26967371

  3. Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

    PubMed Central

    Liang, J.; Edelsbrunner, H.; Woodward, C.

    1998-01-01

    Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et

  4. LISE: a server using ligand-interacting and site-enriched protein triangles for prediction of ligand-binding sites.

    PubMed

    Xie, Zhong-Ru; Liu, Chuan-Kun; Hsiao, Fang-Chih; Yao, Adam; Hwang, Ming-Jing

    2013-07-01

    LISE is a web server for a novel method for predicting small molecule binding sites on proteins. It differs from a number of servers currently available for such predictions in two aspects. First, rather than relying on knowledge of similar protein structures, identification of surface cavities or estimation of binding energy, LISE computes a score by counting geometric motifs extracted from sub-structures of interaction networks connecting protein and ligand atoms. These network motifs take into account spatial and physicochemical properties of ligand-interacting protein surface atoms. Second, LISE has now been more thoroughly tested, as, in addition to the evaluation we previously reported using two commonly used small benchmark test sets and targets of two community-based experiments on ligand-binding site predictions, we now report an evaluation using a large non-redundant data set containing >2000 protein-ligand complexes. This unprecedented test, the largest ever reported to our knowledge, demonstrates LISE's overall accuracy and robustness. Furthermore, we have identified some hard to predict protein classes and provided an estimate of the performance that can be expected from a state-of-the-art binding site prediction server, such as LISE, on a proteome scale. The server is freely available at http://lise.ibms.sinica.edu.tw. PMID:23609546

  5. Ligand-tailored single-site silica supported titanium catalysts: Synthesis, characterization and towards cyanosilylation reaction

    SciTech Connect

    Xu, Wei; Li, Yani; Yu, Bo; Yang, Jindou; Zhang, Ying; Chen, Xi; Zhang, Guofang Gao, Ziwei

    2015-01-15

    A successive anchoring of Ti(NMe{sub 2}){sub 4}, cyclopentadiene and a O-donor ligand, 1-hydroxyethylbenzene (PEA), 1,1′-bi-2-naphthol (Binol) or 2,3-dihydroxybutanedioic acid diethyl ester (Tartrate), on silica was conducted by SOMC strategy in moderate conditions. The silica, monitored by in-situ Fourier transform infrared spectroscopy (in-situ FT-IR), was pretreated at different temperatures (200, 500 and 800 °C). The ligand tailored silica-supported titanium complexes were characterized by in-situ FT-IR, {sup 13}C CP MAS-NMR, X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES) and elemental analysis in detail, verifying that the surface titanium species are single sited. The catalytic activity of the ligand tailored single-site silica supported titanium complexes was evaluated by a cyanosilylation of benzaldehyde. The results showed that the catalytic activity is dependent strongly on the dehydroxylation temperatures of silica and the configuration of the ligands. - Graphical abstract: The ligand-tailored silica supported “single site” titanium complexes were synthesized by SOMC strategy and fully characterized. Their catalytic activity were evaluated by benzaldehyde silylcyanation. - Highlights: • Single-site silica supported Ti active species was prepared by SOMC technique. • O-donor ligand tailored Ti surface species was synthesized. • The surface species was characterized by XPS, {sup 13}C CP-MAS NMR, XANES etc. • Catalytic activity of the Ti active species in silylcyanation reaction was evaluated.

  6. Detection of L-lactate in polyethylene glycol solutions confirms the identity of the active-site ligand in a proline dehydrogenase structure.

    PubMed

    Zhang, Min; Tanner, John J

    2004-05-01

    Polyethylene glycol (PEG) is often used in protein crystallography as a low-ionic-strength precipitant for crystallization and as a cryoprotectant for low-temperature data collection. Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the Escherichia coli PutA proline dehydrogenase domain crystal structure, the L-lactate concentration of several PEG solutions was measured. 50%(w/v) solutions of PEGs with molecular weight 3000, 4000 and 8000 contain millimolar levels of L-lactate. In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350. These results help to explain why L-lactate was present in the proline dehydrogenase domain crystal structure. This work also has implications for the crystallization of enzymes that bind L-lactate. PMID:15103160

  7. The biologically active conformations of ligand CCK B receptor

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Pavel E.; Kuznetsova, Nina B.; Schulgin, Sergey V.; Rogacheva, Svetlana M.; Sinyakov, Valeriy V.; Kovtun, Viktor A.

    2006-07-01

    We analyzed literature data about structures of ligands of CCK B receptor. The structure of the binding site (fragments of the third extracellular loop and the seventh transmembrane helix of CCK B receptor) was determined recently by experiments. We were finding presumable biologically active conformations (BAC) of the ligands by two methods. One of them is based on the fact that the most stable conformations of the biologically active peptide on the phase interface "water-lipophilic medium" are often similar to the BAC. Another method is based on the formation of the stable ligand-receptor complex during the modeling procedure. We used Monte-Carlo method with the fixed geometry of the receptor and the optimized geometry of tetrapeptide cholecystokinin (CCK-4). It has been shown, that the first method should be used to find BAC of antagonists of CCK B receptor. The strategy of the formation of the stable ligand-receptor complex during the modeling procedure can be used for the designing of peptide agonists of CCK B receptor.

  8. Models for the active site in [FeFe] hydrogenase with iron-bound ligands derived from bis-, tris-, and tetrakis(mercaptomethyl)silanes.

    PubMed

    Apfel, Ulf-Peter; Troegel, Dennis; Halpin, Yvonne; Tschierlei, Stefanie; Uhlemann, Ute; Görls, Helmar; Schmitt, Michael; Popp, Jürgen; Dunne, Peter; Venkatesan, Munuswamy; Coey, Michael; Rudolph, Manfred; Vos, Johannes G; Tacke, Reinhold; Weigand, Wolfgang

    2010-11-01

    A series of multifunctional (mercaptomethyl)silanes of the general formula type R(n)Si(CH(2)SH)(4-n) (n = 0-2; R = organyl) was synthesized, starting from the corresponding (chloromethyl)silanes. They were used as multidentate ligands for the conversion of dodecacarbonyltriiron, Fe(3)(CO)(12), into iron carbonyl complexes in which the deprotonated (mercaptomethyl)silanes act as μ-bridging ligands. These complexes can be regarded as models for the [FeFe] hydrogenase. They were characterized by elemental analyses (C, H, S), NMR spectroscopic studies ((1)H, (13)C, (29)Si), and single-crystal X-ray diffraction. Their electrochemical properties were investigated by cyclic voltammetry to disclose a new mechanism for the formation of dihydrogen catalyzed by these compounds, whereby one sulfur atom was protonated in the catalytic cycle. The reaction of the tridentate ligand MeSi(CH(2)SH)(3) with Fe(3)(CO)(12) yielded a tetranuclear cluster compound. A detailed investigation by X-ray diffraction, electrochemical, Raman, Mössbauer, and susceptibility techniques indicates that for this compound initially [Fe(2){μ-MeSi(CH(2)S)(2)CH(2)SH}(CO)(6)] is formed. This dinuclear complex, however, is slowly transformed into the tetranuclear species [Fe(4){μ-MeSi(CH(2)S)(3)}(2)(CO)(8)]. PMID:20873759

  9. Sequence variation in ligand binding sites in proteins

    PubMed Central

    Magliery, Thomas J; Regan, Lynne

    2005-01-01

    Background The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance. Results Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys2His2 zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies. Conclusion Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites. PMID:16194281

  10. Why Is There an “Inert” Metal Center in the Active Site of Nitrile Hydratase? Reactivity and Ligand Dissociation from a Five-Coordinate Co(III) Nitrile Hydratase Model

    PubMed Central

    Shearer, Jason; Kung, Irene Y.; Lovell, Scott; Kaminsky, Werner; Kovacs, Julie A.

    2015-01-01

    To determine how a substitutionally inert metal can play a catalytic role in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate CoIII thiolate complex ([CoIII(S2Me2N3(Pr,Pr))](PF6) (1)) that resembles the active site of cobalt containing nitrile hydratase (Co NHase) was prepared. This was screened for reactivity, by using low-temperature electronic absorption spectroscopy, toward a number of biologically relevant “substrates”. It was determined 1 will react with azide, thiocyanate, and ammonia, but is unreactive toward nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [CoIII(ADIT2)](PF6) (2). Complex 2 is a six-coordinate CoIII complex containing cis-thiolates and imine nitrogens, and has properties similar to the cobalt center of Co NHase. Substrate binding to 1 is reversible and temperature-dependent, allowing for the determination of the thermodynamic parameters of azide and thiocyanate binding and the rates of ligand dissociation. Azide and thiocyanate bind trans to a thiolate, and with similar entropies and enthalpies (thiocyanate: ΔH = −7.5 ± 1.1 kcal/mol, ΔS = −17.2 ± 3.2 eu; azide: ΔH = −6.5 ± 1.0 kcal/mol, ΔS = −12.6 ± 2.4 eu). The rates of azide and thiocyanate displacement from the metal center are also comparable to one another (kd = (7.22 ± 0.04) × 10−1 s−1 for thiocyanate and kd = 2.14 ± 0.50) × 10−2 s−1 for azide), and are considerably faster than one would expect for a low-spin d6 six-coordinate CoIII complex. These rates are comparable to those of an analogous Fe(III) complex, demonstrating that Co(III) and Fe(III) react at comparable rates when in this ligand environment. This study therefore indicates that ligand displacement from a low-spin CoIII center in a ligand environment that resembles NHase is not prohibitivly slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes. PMID:11456548

  11. Supramolecular coordination and antimicrobial activities of constructed mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    El-Sonbati, A. Z.; Diab, M. A.; El-Bindary, A. A.; Abou-Dobara, M. I.; Seyam, H. A.

    2013-03-01

    A novel series of copper(II) and palladium(II) with 4-derivatives benzaldehyde pyrazolone (Ln) were synthesized. The mixed ligand complexes were prepared by using 1,10-phenanthroline (Phen) as second ligand. The structure of these complexes was identified and confirm by elemental analysis, molar conductivity, UV-Vis, IR and 1H NMR spectroscopy and magnetic moment measurements as well as thermal analysis. The ligand behaves as a neutral bidentate ligand through ON donor sites. ESR spectra show the simultaneous presence of a planar trans and a nearly planar cis isomers in the 1:2 ratio for all N,O complexes [Cu(Ln)2]Cl2ṡ2H2O. Schiff bases (Ln) were tested against bacterial species; namely two Gram positive bacteria (Staphylococcus aureus and Bacillus cereus) and two Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) and fungal species (Aspergillus niger, Fusarium oxysporium, Penicillium italicum and Alternaria alternata). The tested compounds have antibacterial activity against S. aureus, B. cereus and K. pneumoniae.

  12. Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase.

    PubMed

    Wang, Yunyi; Ragavan, Mukundan; Hilty, Christian

    2016-05-01

    Protein-ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on (1)H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein (1)H signal predominantly in the methyl group region of the spectra. Using (13)C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein-ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that (13)C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein-ligand interactions. PMID:27189223

  13. Ligand-tailored single-site silica supported titanium catalysts: Synthesis, characterization and towards cyanosilylation reaction

    NASA Astrophysics Data System (ADS)

    Xu, Wei; Li, Yani; Yu, Bo; Yang, Jindou; Zhang, Ying; Chen, Xi; Zhang, Guofang; Gao, Ziwei

    2015-01-01

    A successive anchoring of Ti(NMe2)4, cyclopentadiene and a O-donor ligand, 1-hydroxyethylbenzene (PEA), 1,1‧-bi-2-naphthol (Binol) or 2,3-dihydroxybutanedioic acid diethyl ester (Tartrate), on silica was conducted by SOMC strategy in moderate conditions. The silica, monitored by in-situ Fourier transform infrared spectroscopy (in-situ FT-IR), was pretreated at different temperatures (200, 500 and 800 °C). The ligand tailored silica-supported titanium complexes were characterized by in-situ FT-IR, 13C CP MAS-NMR, X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES) and elemental analysis in detail, verifying that the surface titanium species are single sited. The catalytic activity of the ligand tailored single-site silica supported titanium complexes was evaluated by a cyanosilylation of benzaldehyde. The results showed that the catalytic activity is dependent strongly on the dehydroxylation temperatures of silica and the configuration of the ligands.

  14. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    NASA Astrophysics Data System (ADS)

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  15. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    PubMed Central

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-01-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. PMID:26561004

  16. FOLLITROPIN RECEPTORS CONTAIN CRYPTIC LIGAND BINDING SITES1

    PubMed Central

    Lin, Win; Bernard, Michael P.; Cao, Donghui; Myers, Rebecca V.; Kerrigan, John E.; Moyle, William R.

    2007-01-01

    Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with an FSHR/LHR chimera having only two unique LHR residues similar to the manners in which they dock with LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

  17. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    PubMed Central

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

  18. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  19. The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy.

    PubMed

    Roseboom, Winfried; De Lacey, Antonio L; Fernandez, Victor M; Hatchikian, E Claude; Albracht, Simon P J

    2006-01-01

    In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster

  20. Highly Active Multidentate Ligand-Based Alkyne Metathesis Catalysts.

    PubMed

    Du, Ya; Yang, Haishen; Zhu, Chengpu; Ortiz, Michael; Okochi, Kenji D; Shoemaker, Richard; Jin, Yinghua; Zhang, Wei

    2016-06-01

    Alkyne metathesis catalysts composed of molybdenum(VI) propylidyne and multidentate tris(2-hydroxylbenzyl)methane ligands have been developed, which exhibit excellent stability (remains active in solution for months at room temperature), high activity, and broad functional-group tolerance. The homodimerization and cyclooligomerization of monopropynyl or dipropynyl substrates, including challenging heterocycle substrates (e.g., pyridine), proceed efficiently at 40-55 °C in a closed system. The ligand structure and catalytic activity relationship has been investigated, which shows that the ortho groups of the multidentate phenol ligands are critical to the stability and activity of such a catalyst system. PMID:27113640

  1. Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy.

    PubMed

    Le Sueur, Amanda L; Schaugaard, Richard N; Baik, Mu-Hyun; Thielges, Megan C

    2016-06-01

    The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes. PMID:27164303

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

    PubMed Central

    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/. PMID:25685322

  3. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  4. Mixed ligand complexes of bis(phenylimine) Schiff base ligands incorporating pyridinium moiety. Synthesis, characterization and antibacterial activity

    NASA Astrophysics Data System (ADS)

    Mohamed, Gehad G.; El-Wahab, Zeinab H. Abd

    2005-04-01

    The synthesis and structural characterization of mixed ligand complexes derived from 2,6-pyridinedicarboxaldehydebis( o-hydroxyphenylimine), 2,6-pyridinedicarboxaldehydebis( p-hydroxyphenylimine) (1 ry ligands) and 2-aminopyridne (2 ry ligand) are reported. The ligands and their transition metal complexes were characterized on the bases of their elemental analyses, IR, solid reflectance, magnetic moment, molar conductance and thermal analysis (TGA). The mixed ligand complexes are formed in the 1:1:1 (M:L 1 or L 2:L') ratio as found from the elemental analyses and found to have the formulae [MX 2(L 1 or L 2)(L')]· nH 2O where M = Co(II), Ni(II), Cu(II) and Zn(II), L 1 = 2,6-pyridinedicarboxaldehydebis( p-hydroxyphenylimine), L 2 = 2,6-pyridine dicarboxaldehydebis( o-hydroxyphenylimine), L' = 2-aminopyridine, X = Cl - in case of Cu(II) complex and Br - in case of Co(II), Ni(II) and Zn(II) complexes and y = 0-3. The molar conductance data reveal that the chelates are non-electrolytes. IR spectra show that the Schiff bases are coordinated to the metal ions in a terdentate manner with NNN donor sites of the pyridine- N and two azomethine- N. While 2-aminopyridine coordinated to the metal ions via its pyridine- N. Magnetic and solid reflectance spectra are used to infer the coordinating capacity of the ligand and the geometrical structure of these complexes are found to be octahedral. The thermal behaviour of these chelates shows that the hydrated water molecules and the anions are removed in a successive two steps followed immediately by decomposition of the ligands (L 1, L 2 and L') in the subsequent steps. The activation thermodynamic parameters, such as, E*, Δ H*, Δ S* and Δ G* are calculated from the TG curves and discussed. The ligands and their metal chelates have been screened for their antimicrobial activities and the findings have been reported, explained and compared with some known antibiotics.

  5. Mixed ligand complexes of bis(phenylimine) Schiff base ligands incorporating pyridinium moiety Synthesis, characterization and antibacterial activity.

    PubMed

    Mohamed, Gehad G; Abd El-Wahab, Zeinab H

    2005-04-01

    The synthesis and structural characterization of mixed ligand complexes derived from 2,6-pyridinedicarboxaldehydebis(o-hydroxyphenylimine), 2,6-pyridinedicarboxaldehydebis(p-hydroxyphenylimine) (1(ry) ligands) and 2-aminopyridne (2(ry) ligand) are reported. The ligands and their transition metal complexes were characterized on the bases of their elemental analyses, IR, solid reflectance, magnetic moment, molar conductance and thermal analysis (TGA). The mixed ligand complexes are formed in the 1:1:1 (M:L(1) or L(2):L') ratio as found from the elemental analyses and found to have the formulae [MX(2)(L(1) or L(2))(L')].nH(2)O where M = Co(II), Ni(II), Cu(II) and Zn(II), L(1) = 2,6-pyridinedicarboxaldehydebis(p-hydroxyphenylimine), L(2) = 2,6-pyridine dicarboxaldehydebis(o-hydroxyphenylimine), L' = 2-aminopyridine, X = Cl(-) in case of Cu(II) complex and Br(-) in case of Co(II), Ni(II) and Zn(II) complexes and y = 0-3. The molar conductance data reveal that the chelates are non-electrolytes. IR spectra show that the Schiff bases are coordinated to the metal ions in a terdentate manner with NNN donor sites of the pyridine-N and two azomethine-N. While 2-aminopyridine coordinated to the metal ions via its pyridine-N. Magnetic and solid reflectance spectra are used to infer the coordinating capacity of the ligand and the geometrical structure of these complexes are found to be octahedral. The thermal behaviour of these chelates shows that the hydrated water molecules and the anions are removed in a successive two steps followed immediately by decomposition of the ligands (L(1), L(2) and L') in the subsequent steps. The activation thermodynamic parameters, such as, E*, DeltaH*, DeltaS* and DeltaG* are calculated from the TG curves and discussed. The ligands and their metal chelates have been screened for their antimicrobial activities and the findings have been reported, explained and compared with some known antibiotics. PMID:15741103

  6. Uncoupling the Structure-Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding.

    PubMed

    Dickson, Callum J; Hornak, Viktor; Velez-Vega, Camilo; McKay, Daniel J J; Reilly, John; Sandham, David A; Shaw, Duncan; Fairhurst, Robin A; Charlton, Steven J; Sykes, David A; Pearlstein, Robert A; Duca, Jose S

    2016-06-23

    Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions. PMID:27239696

  7. Crystallographic Studies of the Binding of Ligands to theDicarboxylate Site of Complex II, and the Identity of the Ligand in the'Oxaloacetate-Inhibited' State

    SciTech Connect

    Huang, Li-Shar; Shen, John T.; Wang, Andy C.; Berry, Edward A.

    2006-07-01

    Mitochondrial Complex II (succinate:ubiquinoneoxidoreductase) is purified in a partially innactivated state, which canbe activated by removal of tightly bound oxaloacetate (Kearney, E.B. etal. Biochem Biophys Res Commun 49, 1115-1121). We crystallized Complex IIin the presence of oxaloacetate or with the endogenous inhibitor bound.The structure showed a ligand essentially identical to the "malate-likeintermediate" found in Shewanella Flavocytochrome c crystallized withfumarate (Taylor, P., et al. Nat Struct Biol 6, 1108-1112.)Crystallization of Complex II in the presence of excess fumarate alsogave the malate-like intermediate or a mixture of that and fumarate atthe active site. In order to more conveniently monitor the occupationstate of the dicarboxylate site, we are developing a library of UV/Visspectral effects induced by binding different ligands to the site.Treatment with fumarate results in rapid development of the fumaratedifference spectrum and then a very slow conversion into a speciesspectrally similar to the OAA liganded complex. Complex II is known to becapable of oxidizing malate to the enol form of oxaloacetate (Belikova,Y.O., et al. Biochim Biophys Acta 936, 1-9). The observations abovesuggest it may also be capable of interconverting fumarate and malate. Itmay be useful for understanding the mechanism and regulation of theenzyme to identify the malate-like intermediate and its pathway offormation from oxaloacetate or fumarate.

  8. CRYSTALLOGRAPHIC STUDIES OF THE BINDING OF LIGANDS TO THE DICARBOXYLATE SITE OF COMPLEX II, AND THE IDENTITY OF THE LIGAND IN THE “OXALOACETATE-INHIBITED” STATE.

    PubMed Central

    Huang, Li-Shar; Shen, John T.; Wang, Andy C.; Berry., Edward A.

    2006-01-01

    Summary Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially innactivated state, which can be activated by removal of tightly bound oxaloacetate (Kearney, E. B. et al. Biochem Biophys Res Commun 49, 1115–1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the “malate-like intermediate” found in Shewanella Flavocytochrome c crystallized with fumarate (Taylor, P. et al. Nat Struct Biol 6, 1108–1112.) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Belikova, Y. O. et al. Biochim Biophys Acta 936, 1–9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate. PMID:16935256

  9. Redox-Active-Ligand-Mediated Formation of an Acyclic Trinuclear Ruthenium Complex with Bridging Nitrido Ligands.

    PubMed

    Bagh, Bidraha; Broere, Daniël L J; Siegler, Maxime A; van der Vlugt, Jarl Ivar

    2016-07-11

    Coordination of a redox-active pyridine aminophenol ligand to Ru(II) followed by aerobic oxidation generates two diamagnetic Ru(III) species [1 a (cis) and 1 b (trans)] with ligand-centered radicals. The reaction of 1 a/1 b with excess NaN3 under inert atmosphere resulted in the formation of a rare bis(nitrido)-bridged trinuclear ruthenium complex with two nonlinear asymmetrical Ru-N-Ru fragments. The spontaneous reduction of the ligand centered radical in the parent 1 a/1 b supports the oxidation of a nitride (N(3-) ) to half an equivalent of N2 . The trinuclear omplex is reactive toward TEMPO-H, tin hydrides, thiols, and dihydrogen. PMID:27321547

  10. Surface-mediated synthesis of dimeric rhodium catalysts on MgO: tracking changes in the nuclearity and ligand environment of the catalytically active sites by X-ray absorption and infrared spectroscopies.

    PubMed

    Yardimci, Dicle; Serna, Pedro; Gates, Bruce C

    2013-01-21

    The preparation of dinuclear rhodium clusters and their use as catalysts is challenging because these clusters are unstable, evolving readily into species with higher nuclearities. We now present a novel synthetic route to generate rhodium dimers on the surface of MgO by a stoichiometrically simple surface-mediated reaction involving [Rh(C(2)H(4))(2)] species and H(2). X-ray absorption and IR spectra were used to characterize the changes in the nuclearity of the essentially molecular surface species as they formed, including the ligands on the rhodium and the metal-support interactions. The support plays a key role in stabilizing the dinuclear rhodium species, allowing the incorporation of small ligands (ethyl, hydride, and/or CO) and enabling a characterization of the catalytic performance of the supported species for the hydrogenation of ethylene as a function of the metal nuclearity and ligand environment. A change in the nuclearity from one to two Rh atoms leads to a 58-fold increase in the catalytic activity for ethylene hydrogenation, a reaction involving unsaturated, but stable, dimeric rhodium species. PMID:23208893

  11. DISTINCT ROLES OF β1 MIDAS, ADMIDAS AND LIMBS CATION-BINDING SITES IN LIGAND RECOGNITION BY INTEGRIN α2β1*

    PubMed Central

    Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul

    2012-01-01

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259

  12. Probing heterotrimeric G protein activation: applications to biased ligands

    PubMed Central

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands. PMID:22229559

  13. Probing heterotrimeric G protein activation: applications to biased ligands.

    PubMed

    Denis, Colette; Saulière, Aude; Galandrin, Segolene; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands. PMID:22229559

  14. Proteus and the Design of Ligand Binding Sites.

    PubMed

    Polydorides, Savvas; Michael, Eleni; Mignon, David; Druart, Karen; Archontis, Georgios; Simonson, Thomas

    2016-01-01

    This chapter describes the organization and use of Proteus, a multitool computational suite for the optimization of protein and ligand conformations and sequences, and the calculation of pK α shifts and relative binding affinities. The software offers the use of several molecular mechanics force fields and solvent models, including two generalized Born variants, and a large range of scoring functions, which can combine protein stability, ligand affinity, and ligand specificity terms, for positive and negative design. We present in detail the steps for structure preparation, system setup, construction of the interaction energy matrix, protein sequence and structure optimizations, pK α calculations, and ligand titration calculations. We discuss illustrative examples, including the chemical/structural optimization of a complex between the MHC class II protein HLA-DQ8 and the vinculin epitope, and the chemical optimization of the compstatin analog Ac-Val4Trp/His9Ala, which regulates the function of protein C3 of the complement system. PMID:27094287

  15. Effect of Bridgehead Steric Bulk on the Intramolecular C-H Heterolysis of [FeFe]-Hydrogenase Active Site Models Containing a P2N2 Pendant Amine Ligand.

    PubMed

    Zheng, Dehua; Wang, Mei; Wang, Ning; Cheng, Minglun; Sun, Licheng

    2016-01-19

    A series of pendant amine-containing [FeFe]-hydrogenase models, [X(CH2S-μ)2{Fe(CO)3}{Fe(CO)(P2(Ph)N2(Bn))}] (1H, X = CH2; 2Me, C(CH3)2; 3Et, C(CH2CH3)2; and P2(Ph)N2(Bn) = 1,5-dibenzyl-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane) with different groups at the bridgehead carbon of the S-to-S linker were synthesized. The oxidations of these complexes as well as the reverse reduction reaction were studied by cyclic voltammetry and in situ IR spectroscopy. Regardless of the bridgehead steric bulk, all three complexes demonstrate intramolecular iron-mediated C(sp(3))-H bond heterolytic cleavage with the assistance of the pendant amine base within the chelating diphosphine ligand in the two-electron oxidation process. X-ray crystallographic analysis shows that the doubly oxidized products, [1'H](+), [2'Me](+), and [3'Et](+), all have a rigid FeSC three-membered ring at the open apical site of the rotated iron center. The most noticeable difference in structures of the oxidized complexes is that the single CO ligand of the rotated Fe(P2(Ph)N2(Bn))(CO) unit in [1'H](+) and [2'Me](+) is found below the Fe···Fe vector, while in [3'Et](+) an unusually rotated Fe(P2(Ph)N2(Bn))(CO) moiety positions one of the P donors within the bidentate ligand under the Fe···Fe vector. The starting Fe(I)Fe(I) complexes can be recovered from their corresponding doubly oxidized complexes by reduction in the presence of Brönsted acid. PMID:26230977

  16. A Guided Inquiry Activity for Teaching Ligand Field Theory

    ERIC Educational Resources Information Center

    Johnson, Brian J.; Graham, Kate J.

    2015-01-01

    This paper will describe a guided inquiry activity for teaching ligand field theory. Previous research suggests the guided inquiry approach is highly effective for student learning. This activity familiarizes students with the key concepts of molecular orbital theory applied to coordination complexes. Students will learn to identify factors that…

  17. Chiroptical activity in colloidal quantum dots coated with achiral ligands.

    PubMed

    Melnikau, Dzmitry; Savateeva, Diana; Gaponik, Nikolai; Govorov, Alexander O; Rakovich, Yury P

    2016-01-25

    We studied the chiroptical properties of colloidal solution of CdSe and CdSe/ZnS quantum dots (QDs) with a cubic lattice structure which were initially prepared without use of any chiral molecules and coated with achiral ligands. We demonstrate circular dichroism (CD) activity around first and second excitonic transition of these CdSe based nanocrystals. We consider that this chiroptical activity is caused by imbalance in racemic mixtures of QDs between the left and right handed nanoparticles, which appears as a result of the formation of various defects or incorporation of impurities into crystallographic structure during their synthesis. We demonstrate that optical activity of colloidal solution of CdSe QDs with achiral ligands weakly depends on the QDs size and number of ZnS monolayers, but does not depend on the nature of achiral ligands or polarity of the solution. PMID:26832599

  18. The Potential Applications of Peroxisome Proliferator-Activated Receptor δ Ligands in Assisted Reproductive Technology

    PubMed Central

    Huang, Jaou-Chen

    2008-01-01

    Peroxisome proliferator-activated receptor δ (PPARδ, also known as PPARβ) has ubiquitous distribution and extensive biological functions. The reproductive function of PPARδ was first revealed in the uterus at the implantation site. Since then, PPARδ and its ligand have been discovered in all reproductive tissues, including the gametes and the preimplantation embryos. PPARδ in preimplantation embryos is normally activated by oviduct-derived PPARδ ligand. PPARδ activation is associated with an increase in embryonic cell proliferation and a decrease in programmed cell death (apoptosis). On the other hand, the role of PPARδ and its ligand in gamete formation and function is less well understood. This review will summarize the reproductive functions of PPARδ and project its potential applications in assisted reproductive technology. PMID:19096716

  19. A single ligand is sufficient to activate EGFR dimers

    PubMed Central

    Liu, Ping; Cleveland, Thomas E.; Bouyain, Samuel; Byrne, Patrick O.; Longo, Patti A.; Leahy, Daniel J.

    2012-01-01

    Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions. PMID:22699492

  20. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes. PMID:17168764

  1. In silico Identification and Characterization of Protein-Ligand Binding Sites.

    PubMed

    Roche, Daniel Barry; McGuffin, Liam James

    2016-01-01

    Protein-ligand binding site prediction methods aim to predict, from amino acid sequence, protein-ligand interactions, putative ligands, and ligand binding site residues using either sequence information, structural information, or a combination of both. In silico characterization of protein-ligand interactions has become extremely important to help determine a protein's functionality, as in vivo-based functional elucidation is unable to keep pace with the current growth of sequence databases. Additionally, in vitro biochemical functional elucidation is time-consuming, costly, and may not be feasible for large-scale analysis, such as drug discovery. Thus, in silico prediction of protein-ligand interactions must be utilized to aid in functional elucidation. Here, we briefly discuss protein function prediction, prediction of protein-ligand interactions, the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated EvaluatiOn (CAMEO) competitions, along with their role in shaping the field. We also discuss, in detail, our cutting-edge web-server method, FunFOLD for the structurally informed prediction of protein-ligand interactions. Furthermore, we provide a step-by-step guide on using the FunFOLD web server and FunFOLD3 downloadable application, along with some real world examples, where the FunFOLD methods have been used to aid functional elucidation. PMID:27094282

  2. Identification of co-evolving sites in the ligand binding domain of G protein-coupled receptors using mutual information

    NASA Astrophysics Data System (ADS)

    Fatakia, Sarosh N.; Costanzi, Stefano; Chow, Carson C.

    2008-03-01

    G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in humans. They are involved in signal transduction in numerous cellular processes and are the most common target for pharmacological intervention via activation or inhibition. Identification of functionally important sites is relevant for better understanding the ligand-receptor interaction and therefore for drug delivery. In a superfamily of proteins, functionally important but co-evolving sites are not easily identified in a multiple sequence alignment (MSA). Using a MSA of trans-membrane (TM) domains of GPCR superfamily, we identify sites which co-evolve, and may therefore be functionally important. Assigning the TM site as a node and the MI of site pairs as an inverse inter-node distance, a MI graph is established. Co-evolving sites are then identified via this graph. Nodes characterized by high connectivity are located within the commonly accepted ligand binding site of GPCRs, suggesting that concerted co-evolution of a number of neighboring residues gave rise to a multitude of subfamilies each recognizing a specific set of ligands. MI and graph analysis may serve as a tool for the identification of topologically conserved binding pockets in the families of evolutionarily related proteins.

  3. Ligands for the Nuclear Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Sauer, Sascha

    2015-10-01

    Nuclear receptors are ligand-activated transcription factors, which represent a primary class of drug targets. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a key player in various biological processes. PPARγ is widely known as the target protein of the thiazolidinediones for treating type 2 diabetes. Moreover, PPARγ ligands can induce anti-inflammatory and potentially additional beneficial effects. Recent mechanistic insights of PPARγ modulation give hope the next generation of efficient PPARγ-based drugs with fewer side effects can be developed. Furthermore, chemical approaches that make use of synergistic action of combinatorial ligands are promising alternatives for providing tailored medicine. Lessons learned from fine-tuning the action of PPARγ can provide avenues for efficient molecular intervention via many other nuclear receptors to combat common diseases. PMID:26435213

  4. The good, the bad and the dubious: VHELIBS, a validation helper for ligands and binding sites

    PubMed Central

    2013-01-01

    Background Many Protein Data Bank (PDB) users assume that the deposited structural models are of high quality but forget that these models are derived from the interpretation of experimental data. The accuracy of atom coordinates is not homogeneous between models or throughout the same model. To avoid basing a research project on a flawed model, we present a tool for assessing the quality of ligands and binding sites in crystallographic models from the PDB. Results The Validation HElper for LIgands and Binding Sites (VHELIBS) is software that aims to ease the validation of binding site and ligand coordinates for non-crystallographers (i.e., users with little or no crystallography knowledge). Using a convenient graphical user interface, it allows one to check how ligand and binding site coordinates fit to the electron density map. VHELIBS can use models from either the PDB or the PDB_REDO databank of re-refined and re-built crystallographic models. The user can specify threshold values for a series of properties related to the fit of coordinates to electron density (Real Space R, Real Space Correlation Coefficient and average occupancy are used by default). VHELIBS will automatically classify residues and ligands as Good, Dubious or Bad based on the specified limits. The user is also able to visually check the quality of the fit of residues and ligands to the electron density map and reclassify them if needed. Conclusions VHELIBS allows inexperienced users to examine the binding site and the ligand coordinates in relation to the experimental data. This is an important step to evaluate models for their fitness for drug discovery purposes such as structure-based pharmacophore development and protein-ligand docking experiments. PMID:23895374

  5. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  6. Removal and recovery of toxic metal ions from aqueous waste sites using polymer pendant ligands

    SciTech Connect

    Fish, D.

    1996-10-01

    The purpose of this project is to investigate the use of polymer pendant ligand technology to remove and recover toxic metal ions from DOE aqueous waste sites. Polymer pendant lgiands are organic ligands, anchored to crosslinked, modified divinylbenzene-polystyrene beads, that can selectively complex metal ions. The metal ion removal step usually occurs through a complexation or ion exchange phenomena, thus recovery of the metal ions and reuse of the beads is readily accomplished.

  7. Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

    PubMed Central

    Bohnuud, Tanggis; Kozakov, Dima; Vajda, Sandor

    2014-01-01

    Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although

  8. Intracavitary ligand distribution in tear lipocalin by site-directed tryptophan fluorescence.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2009-08-01

    Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding. PMID:19586017

  9. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

    PubMed

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  10. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel

    PubMed Central

    Joseph, Thomas T.

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  11. Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites

    PubMed Central

    Singh, Rajiv Ranjan; Mishra, Satyendra; Gupta, Sarika; Surolia, Avadhesha; Salunke, Dinakar M.

    2012-01-01

    Background Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4) and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis. Methodology and Principal Findings We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site) than the second T4 site (BD site). Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy. Conclusion Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation. PMID:22973437

  12. Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    PubMed Central

    Fritsch, Cornelia; Klaver, Arjen; Kanger, Johannes S.; Jovin, Thomas M.; Arndt-Jovin, Donna J.

    2013-01-01

    Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor

  13. A structural view of ligand-dependent activation in thermoTRP channels

    PubMed Central

    Steinberg, Ximena; Lespay-Rebolledo, Carolyne; Brauchi, Sebastian

    2014-01-01

    Transient Receptor Potential (TRP) proteins are a large family of ion channels, grouped into seven sub-families. Although great advances have been made regarding the activation and modulation of TRP channel activity, detailed molecular mechanisms governing TRP channel gating are still needed. Sensitive to electric, chemical, mechanical, and thermal cues, TRP channels are tightly associated with the detection and integration of sensory input, emerging as a model to study the polymodal activation of ion channel proteins. Among TRP channels, the temperature-activated kind constitute a subgroup by itself, formed by Vanilloid receptors 1–4, Melastatin receptors 2, 4, 5, and 8, TRPC5, and TRPA1. Some of the so-called “thermoTRP” channels participate in the detection of noxious stimuli making them an interesting pharmacological target for the treatment of pain. However, the poor specificity of the compounds available in the market represents an important obstacle to overcome. Understanding the molecular mechanics underlying ligand-dependent modulation of TRP channels may help with the rational design of novel synthetic analgesics. The present review focuses on the structural basis of ligand-dependent activation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection of ligand-binding sites within TRPV1, PIP2-dependent modulation of TRP channels, and the structure of natural and synthetic ligands. PMID:24847275

  14. Triphenylethylene antiestrogen-binding sites in cockerel liver nuclei: evidence for an endogenous ligand.

    PubMed

    Murphy, P R; Butts, C; Lazier, C B

    1984-07-01

    Salt extracts of purified nuclei from cockerel liver contain a limited number of sites that bind triphenylethylene nonsteroidal antiestrogens with high affinity and specificity. The assay of the [3H]tamoxifen (3H-labeled 1-[4-(2-dimethylaminoethyoxy)phenyl] 1,2-diphenylbut-1-(Z)ene)-binding sites is optimally achieved by preincubation of the salt extracts with charcoal-dextran suspension; a 4- to 8-fold increase in activity over that obtained with nontreated extracts is found. This suggests that the binding sites are occupied in vivo by an unknown endogenous ligand. The equilibrium dissociation constant for [3H]tamoxifen binding is 4.76 +/- 1.8 nM, and the binding site concentration is 1.7 +/- 0.7 fmol/microgram DNA. The concentration of high affinity estrogen-binding sites in the same extracts is almost 30-fold less (0.06 +/- 0.01 fmol/micrograms DNA). The relative binding affinities of various antiestrogens for the nuclear antiestrogen-binding sites (with tamoxifen arbitrarily set at 100%) are as follows: nafoxidine (1-[2-(p-[3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl]phenoxy)ethyl] pyrrolidine hydrochloride); 126%) greater than tamoxifen (100%) greater than N-des-methyltamoxifen (16%) greater than CI-628 (alpha-[p-[2-(1-pyrrolidine)ethyoxy]phenyl] 4-methoxy-alpha'-nitrostilbene; 14%) greater than 4-hydroxytamoxifen (7%). Estrogens (17 beta-estradiol, estriol, estrone, and diethylstilbestrol) and several other steroids (cholesterol, dihydrotestosterone, pregnenolone, progesterone, and hydrocortisone) show little or no affinity for binding to the nuclear sites (relative binding affinity, less than 0.5%). However, ether extracts of cockerel serum or liver nuclei contain a substance(s) that competitively inhibits [3H]tamoxifen binding to the nuclear antiestrogen-binding sites. The ether-soluble material does not compete for [3H]estradiol binding to the salt-soluble nuclear estrogen receptor. These studies suggest that cockerel serum and liver nuclei contain a natural

  15. Ligand Mobility Modulates Immunological Synapse Formation and T Cell Activation

    PubMed Central

    Hsu, Chih-Jung; Hsieh, Wan-Ting; Waldman, Abraham; Clarke, Fiona; Huseby, Eric S.; Burkhardt, Janis K.; Baumgart, Tobias

    2012-01-01

    T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca2+ were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses. PMID:22384241

  16. Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

    PubMed Central

    Pineda-Molina, Estela; Reyes-Darias, José-Antonio; Lacal, Jesús; Ramos, Juan L.; García-Ruiz, Juan Manuel; Gavira, Jose A.; Krell, Tino

    2012-01-01

    Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate. PMID:23112148

  17. Ligand binding sites of Na,K-ATPase.

    PubMed

    Lingrel, J B; Croyle, M L; Woo, A L; Argüello, J M

    1998-08-01

    Our studies have concentrated on two aspects of the Na,K-ATPase, the first relates to the identification of amino acids involved in binding Na+ and K+ during the catalytic cycle and the second involves defining how cardiac glycosides inhibit the enzyme. To date, three amino acids, Ser775, Asp804 and Asp808, all located in transmembrane regions five and six, have been shown to play a major role in K+ binding. These findings are based on site directed mutagenesis and expression studies. In order to understand how cardiac glycosides interact with the Na,K-ATPase, studies again involving mutagenesis coupled with expression have been used. More specifically, amino acid residues have been substituted in an ouabain sensitive alpha subunit using random mutagenesis, and the ability of the resulting enzyme to confer resistance to ouabain sensitive cells was determined. Interestingly, the amino acids of the alpha subunit which alter ouabain sensitivity cluster in two major regions, one comprised of the first and second transmembrane spanning domains and the extracellular loop joining them, and the second formed by the extracellular halves of transmembrane regions four, five, six and seven. As noted above, transmembrane regions five and six also contain the three amino acid residues Ser775, Asp804 and Asp808 which play a key role in cation transport, possibly binding K+. Thus, it is reasonable to propose that cardiac glycosides bind to two sites, the N- terminal region and the central region which contains the cation binding sites. Cardiac glycoside binding to the center region may lock the cation transport region into a configuration such that the enzyme cannot go through the conformational change required for ion transport. PMID:9789548

  18. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  19. Structural insights into ligand-induced activation of the insulin receptor

    SciTech Connect

    Ward, C.; Lawrence, M.; Streltsov, V.; Garrett, T.; McKern, N.; Lou, M.-Z.; Lovrecz, G.; Adams, T.

    2008-04-29

    The current model for insulin binding to the insulin receptor proposes that there are two binding sites, referred to as sites 1 and 2, on each monomer in the receptor homodimer and two binding surfaces on insulin, one involving residues predominantly from the dimerization face of insulin (the classical binding surface) and the other residues from the hexamerization face. High-affinity binding involves one insulin molecule using its two surfaces to make bridging contacts with site 1 from one receptor monomer and site 2 from the other. Whilst the receptor dimer has two identical site 1-site 2 pairs, insulin molecules cannot bridge both pairs simultaneously. Our structures of the insulin receptor (IR) ectodomain dimer and the L1-CR-L2 fragments of IR and insulin-like growth factor receptor (IGF-1R) explain many of the features of ligand-receptor binding and allow the two binding sites on the receptor to be described. The IR dimer has an unexpected folded-over conformation which places the C-terminal surface of the first fibronectin-III domain in close juxtaposition to the known L1 domain ligand-binding surface suggesting that the C-terminal surface of FnIII-1 is the second binding site involved in high-affinity binding. This is very different from previous models based on three-dimensional reconstruction from scanning transmission electron micrographs. Our single-molecule images indicate that IGF-1R has a morphology similar to that of IR. In addition, the structures of the first three domains (L1-CR-L2) of the IR and IGF-1R show that there are major differences in the two regions governing ligand specificity. The implications of these findings for ligand-induced receptor activation will be discussed. This review summarizes the key findings regarding the discovery and characterization of the insulin receptor, the identification and arrangement of its structural domains in the sequence and the key features associated with ligand binding. The remainder of the review

  20. Metal-ligand cooperation in H2 activation with iron complexes bearing hemilabile bis(diphenylphosphino)amine ligands.

    PubMed

    Frank, Nicolas; Hanau, Katharina; Langer, Robert

    2014-10-20

    The octahedral transition-metal complex [(dppa)Fe(Ph2P-N-PPh2)2] (1) [dppa = bis(diphenylphosphino)amine] with homofunctional bidentate ligands is described. The ligand exhibits hemilability due to its small bite angle and the steric repulsion of the coordinated donor groups. As the {Ph2P-N-PPh2}(-) ligand can act as an internal base, heterolytic cleavage of dihydrogen by complex 1 leads to the formation of the hydride complex [(dppa)(Ph2P-N-PPh2)Fe(H)(κ(1)-Ph2P-NH-PPh2)2] (2), representing an example of cooperative bond activation with a homofunctional hemilabile ligand. This study demonstrates that hemilability of homofunctionalized ligands can be affected by careful adjustment of geometric parameters. PMID:25290535

  1. Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

    NASA Astrophysics Data System (ADS)

    Sampath, K.; Sathiyaraj, S.; Raja, G.; Jayabalakrishnan, C.

    2013-08-01

    The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and spectroscopic methods. The title compounds act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  2. Ligand binding and proton exchange dynamics in site-specific mutants of human myoglobin

    SciTech Connect

    Lambright, D.G.

    1992-01-01

    Site specific mutagenesis was used to make substitutions of four residues in the distal heme pocket of human myoglobin: Val68, His64, Lys45, and Asp60. Strongly diffracting crystals of the conservative mutation K45R in the met aquo form were grown in the trigonal space group P3[sub 2]21 and the X-ray crystal structure determined at 1.6 [angstrom] resolution. The overall structure is similar to that of sperm whale met aquo myoglobin. Several of the mutant proteins were characterized by 2-D NMR spectroscopy. The NMR data suggest the structural changes are localized to the region of the mutation. The dynamics of ligand binding to myoglobin mutants were studied by transient absorption spectroscopy following photolysis of the CO complexes. Transient absorption kinetics and spectra on the ns to ms timescale were measured in aqueous solution from 280 K to 310 K and in 75% glycerol: water from 250 K to 310 K. Two significant basis spectra were obtained from singular value decomposition of the matrix of time dependent spectra. The information was used to obtain approximations for the extent of ligand rebinding and the kinetics of conformational relaxation. Except for K45R, substitutions at Lys45 or Asp60 produce changes in the kinetics for ligand rebinding. Replacement of Lys45 with Arg increases the rate of ligand rebinding from the protein matrix by a factor of 2, but does not alter the rates for ligand escape or entry into the protein or the dynamics of the conformational relaxation. Substitutions at His64 and Val68 influence the kinetics of ligand rebinding and the dynamics of conformational relaxation. The results do not support the hypothesis that ligand migration between the heme pocket and solvent is determined solely by fluctuations of Arg45 and His64 between open and closed conformations of the heme pocket but can be rationalized if ligand diffusion through the protein matrix involves multiple competing pathways.

  3. New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster.

    PubMed

    Jeon, Won Bae; Singer, Steven W; Ludden, Paul W; Rubio, Luis M

    2005-12-01

    Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH*Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step. PMID:16283394

  4. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed Central

    Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.

    2000-01-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  5. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2000-02-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  6. Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation.

    PubMed

    Cieniewicz, Anne M; Cooper, Philip R; McGehee, Jennifer; Lingham, Russell B; Kihm, Anthony J

    2016-08-01

    Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize

  7. Inclusion of multiple fragment types in the site identification by ligand competitive saturation (SILCS) approach.

    PubMed

    Raman, E Prabhu; Yu, Wenbo; Lakkaraju, Sirish K; MacKerell, Alexander D

    2013-12-23

    The site identification by ligand competitive saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing molecular dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced ligand grid free energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show that some but not all experimental trends are predicted and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed. PMID:24245913

  8. Inclusion of multiple fragment types in the Site Identification by Ligand Competitive Saturation (SILCS) approach

    PubMed Central

    Raman, E. Prabhu; Yu, Wenbo; Lakkaraju, Sirish K.; MacKerell, Alexander D.

    2014-01-01

    The Site Identification by Ligand Competitive Saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing Molecular Dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors, and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple, diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced Ligand Grid Free Energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show some, but not all experimental trends are predicted, and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed. PMID:24245913

  9. Predicting ligand binding affinity with alchemical free energy methods in a polar model binding site

    PubMed Central

    Boyce, Sarah E.; Mobley, David L.; Rocklin, Gabriel; Graves, Alan P.

    2009-01-01

    We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed relative binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 (phenol) and 1.1 (catechol) kcal/mol. To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0Å (diverse set), 1.8Å (phenol derived predictions) and 1.2Å (catechol derived predictions). We found that to predict accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe these results can help guide future improvements in physics-based absolute binding free energy methods. PMID:19782087

  10. Design and Synthesis of Photoaffinity Labeling Ligands of the l-Prolyl-l-leucyl-glycinmide Binding Site Involved in the Allosteric Modulation of the Dopamine Receptor

    PubMed Central

    Fisher, Abigail; Mann, Amandeep; Verma, Vaneeta; Thomas, Nancy; Mishra, Ram K.; Johnson, Rodney L.

    2008-01-01

    Pro-Leu-Gly-NH2 (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D2 receptors within the CNS. However, the precise binding site of PLG is unknown. Potential photoaffinity labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity labeling ligands were designed and synthesized based upon γ-lactam PLG peptidomimetic 1. The 4-azido-benzoyl and 4-azido-2-hydroxy-benzoyl photoaffinity labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [3H]N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity labeling agents are binding at the same site as PLG and PLG peptidomimetic 1. PMID:16392815

  11. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  12. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  13. Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

    PubMed Central

    Smith, Rachel N; Gonzales, Eric B

    2014-01-01

    Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli. PMID:24262969

  14. Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage.

    PubMed

    Garcia-Vallvé, Santiago; Guasch, Laura; Tomas-Hernández, Sarah; del Bas, Josep Maria; Ollendorff, Vincent; Arola, Lluís; Pujadas, Gerard; Mulero, Miquel

    2015-07-23

    Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs. PMID:25734377

  15. Mono- and binuclear copper(II) complexes of new hydrazone ligands derived from 4,6-diacetylresorcinol: Synthesis, spectral studies and antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Shebl, Magdy; El-ghamry, Mosad A.; Khalil, Saied M. E.; Kishk, Mona A. A.

    Two new hydrazone ligands, H2L1 and H2L2, were synthesized by the condensation of 4,6-diacetylresorcinol with 3-hydrazino-5,6-diphenyl-1,2,4-triazine and isatin monohydrazone, respectively. The structures of the ligands were elucidated by elemental analyses, IR, 1H NMR, electronic and mass spectra. Reactions of the ligands with several copper(II) salts, including AcO-, NO3-, SO42-, Cl- and Br- afforded mono- and binuclear metal complexes. Also, the ligands were allowed to react with Cu(II) ion in the presence of a secondary ligand (L‧) [N,O-donor; 8-hydroxyquinoline, N,N-donor; 1,10-phenanthroline or O,O-donor; benzoylacetone]. Characterization and structure elucidation of the prepared complexes were achieved by elemental and thermal analyses, IR, electronic, mass and ESR spectra as well as conductivity and magnetic susceptibility measurements. The ESR spin Hamiltonian parameters of some complexes were calculated. The spectroscopic data showed that the H2L1 ligand acts as a neutral or monobasic tridentate ligand while the H2L2 ligand acts as a bis(monobasic tridentate) ligand. The coordination sites with the copper(II) ion are phenolic oxygen, azomethine nitrogen and triazinic nitrogen (H2L1 ligand) or isatinic oxygen (H2L2 ligand). The metal complexes exhibited octahedral and square planar geometrical arrangements depending on the nature of the anion. The ligands and some metal complexes showed antimicrobial activity.

  16. Identification of Ligand Binding Sites of Proteins Using the Gaussian Network Model

    PubMed Central

    Tuzmen, Ceren; Erman, Burak

    2011-01-01

    The nonlocal nature of the protein-ligand binding problem is investigated via the Gaussian Network Model with which the residues lying along interaction pathways in a protein and the residues at the binding site are predicted. The predictions of the binding site residues are verified by using several benchmark systems where the topology of the unbound protein and the bound protein-ligand complex are known. Predictions are made on the unbound protein. Agreement of results with the bound complexes indicates that the information for binding resides in the unbound protein. Cliques that consist of three or more residues that are far apart along the primary structure but are in contact in the folded structure are shown to be important determinants of the binding problem. Comparison with known structures shows that the predictive capability of the method is significant. PMID:21283550

  17. Structure-Activity Relationships of Constrained Phenylethylamine Ligands for the Serotonin 5-HT2 Receptors

    PubMed Central

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L.; Gloriam, David E.

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9–11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9–11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9–11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands. PMID:24244317

  18. Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

    PubMed

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-01-01

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems. PMID:26694353

  19. Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

    PubMed

    DeHaven-Hudkins, D L; Ford-Rice, F Y; Allen, J T; Hudkins, R L

    1993-01-01

    The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo. PMID:8515681

  20. Two disparate ligand binding sites in the human P2Y1 receptor

    PubMed Central

    Zhang, Dandan; Gao, Zhan-Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

    2015-01-01

    In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. PMID:25822790

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

    PubMed Central

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

    2016-01-01

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

  2. Proteins and Their Interacting Partners: An Introduction to Protein–Ligand Binding Site Prediction Methods

    PubMed Central

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-01-01

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems. PMID:26694353

  3. Metal-Metal Interactions in Heterobimetallic Complexes with Dinucleating Redox-Active Ligands.

    PubMed

    Broere, Daniël L J; Modder, Dieuwertje K; Blokker, Eva; Siegler, Maxime A; van der Vlugt, Jarl Ivar

    2016-02-12

    The tuning of metal-metal interactions in multinuclear assemblies is a challenge. Selective P coordination of a redox-active PNO ligand to Au(I) followed by homoleptic metalation of the NO pocket with Ni(II) affords a unique trinuclear Au-Ni-Au complex. This species features two antiferromagnetically coupled ligand-centered radicals and a double intramolecular d(8)-d(10) interaction, as supported by spectroscopic, single-crystal X-ray diffraction, and computational data. A corresponding cationic dinuclear Au-Ni analogue with a stronger d(8)-d(10) interaction is also reported. Although both heterobimetallic structures display rich electrochemistry, only the trinuclear Au-Ni-Au complex facilitates electrocatalytic C-X bond activation of alkyl halides in its doubly reduced state. Hence, the presence of a redox-active ligand framework, an available coordination site at gold, and the nature of the nickel-gold interaction appear to be essential for this reactivity. PMID:26762546

  4. Ligand and structure-based approaches for the identification of SIRT1 activators.

    PubMed

    Vyas, Vivek K; Goel, Ashutosh; Ghate, Manjunath; Patel, Palak

    2015-02-25

    SIRT1 is a NAD(+)-dependent deacetylase that involved in various important metabolic pathways. Combined ligand and structure-based approach was utilized for identification of SIRT1 activators. Pharmacophore models were developed using DISCOtech and refined with GASP module of Sybyl X software. Pharmacophore models were composed of two hydrogen bond acceptor (HBA) atoms, two hydrogen bond donor (HBD) sites and one hydrophobic (HY) feature. The pharmacophore models were validated through receiver operating characteristic (ROC) and Güner-Henry (GH) scoring methods. Model-2 was selected as best model among the model 1-3, based on ROC and GH score value, and found reliable in identification of SIRT1 activators. Model-2 (3D search query) was searched against Zinc database. Several compounds with different chemical scaffold were retrieved as hits. Currently, there is no experimental SIRT1 3D structure available, therefore, we modeled SIRT1 protein structure using homology modeling. Compounds with Qfit value of more than 86 were selected for docking study into the SIRT1 homology model to explore the binding mode of retrieved hits in the active allosteric site. Finally, in silico ADMET prediction study was performed with two best docked compounds. Combination of ligand and structure-based modeling methods identified active hits, which may be good lead compounds to develop novel SIRT1 activators. PMID:25595223

  5. Sigma ligand S14905 and locomotor activity in mice.

    PubMed

    Hascoet, M; Bourin, M; Payeur, R; Lombet, A; Peglion, J L

    1995-12-01

    The binding and locomotor profile of a new sigma ligand, S14905, (isobutyl-N-(1-indan-2yl-piperid-4-yl)N-methyl carbamate, furamate) was studied. The binding data revealed that S14905 has a high affinity for sigma receptors and very low affinity for both dopamine D1 and D2 receptors. We have demonstrated that this sigma ligand prevents the locomotor stimulation induced by morphine (32 and 64 mg/kg), cocaine (16 mg/kg), amphetamine (4 mg/kg) and adrafinil (32 mg/kg) at doses lower than those required to depress spontaneous locomotor activity. The antagonism observed in the present study seems to be more specific of morphine induced hyperlocomotion. The high affinity of this compound for sigma receptors makes it a good choice to study the role of this receptor in the CNS. In addition, S14905 does not directly block dopamine receptors but may modulate them in some manner, and would thus warrant further study as a potential atypical antipsychotic agent, and an antagonist for the hyperactivity induced by opiate drug. PMID:8998401

  6. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  7. Simple Ligand-Receptor Interaction Descriptor (SILIRID) for alignment-free binding site comparison.

    PubMed

    Chupakhin, Vladimir; Marcou, Gilles; Gaspar, Helena; Varnek, Alexandre

    2014-06-01

    We describe SILIRID (Simple Ligand-Receptor Interaction Descriptor), a novel fixed size descriptor characterizing protein-ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs) by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor) and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion). Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein-ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91). SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM): sc-PDB SILIRID data form clusters corresponding to different protein types. PMID:25210596

  8. LIBSA – A Method for the Determination of Ligand-Binding Preference to Allosteric Sites on Receptor Ensembles

    PubMed Central

    2015-01-01

    Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor. PMID:24437606

  9. Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design.

    PubMed

    Tan, Yaw Sing; Reeks, Judith; Brown, Christopher J; Thean, Dawn; Ferrer Gago, Fernando Jose; Yuen, Tsz Ying; Goh, Eunice Tze Leng; Lee, Xue Er Cheryl; Jennings, Claire E; Joseph, Thomas L; Lakshminarayanan, Rajamani; Lane, David P; Noble, Martin E M; Verma, Chandra S

    2016-09-01

    Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design. PMID:27532490

  10. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

    PubMed Central

    Richter, Lars; de Graaf, Chris; Sieghart, Werner; Varagic, Zdravko; Mörzinger, Martina; de Esch, Iwan J P; Ecker, Gerhard F; Ernst, Margot

    2012-01-01

    Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. PMID:22446838

  11. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    PubMed Central

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  12. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling. PMID:25420878

  13. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

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

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    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. PMID:26506102

  15. Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

    PubMed Central

    Gready, J. E.; Ranganathan, S.; Schofield, P. R.; Matsuo, Y.; Nishikawa, K.

    1997-01-01

    Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested. PMID:9144769

  16. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  17. Novel ligands rationally designed for characterizing I2-imidazoline binding sites nature and functions.

    PubMed

    Gentili, Francesco; Cardinaletti, Claudia; Vesprini, Cristian; Ghelfi, Francesca; Farande, Aniket; Giannella, Mario; Piergentili, Alessandro; Quaglia, Wilma; Mattioli, Laura; Perfumi, Marina; Hudson, Alan; Pigini, Maria

    2008-08-28

    The study of two series of 2-aryl-ethylen-imidazolines 3-7 and 8-12 inspired by I2-IBS ligands phenyzoline (1) and diphenyzoline (2), respectively, confirmed the interesting "positive" or "negative" morphine analgesia modulation displayed by their corresponding leads and demonstrated that these effects might be correlated with morphine tolerance and dependence, respectively. By comparative examination of rationally designed compounds, some analogies between binding site cavity of I2-IBS proteins and alpha 2C-adrenoreceptor emerged. PMID:18661965

  18. 3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors.

    PubMed

    Filizola, M; Laakkonen, L; Loew, G H

    1999-11-01

    Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently

  19. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    SciTech Connect

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  20. Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine-based affinity ligands.

    PubMed

    Lin, Chih-Pei; Boysen, Reinhard I; Campi, Eva M; Saito, Kei; Hearn, Milton T W

    2016-07-01

    This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine-based ligands immobilized onto agarose-derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2-ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine-based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26842829

  1. Rational design of a protein that binds integrin αvβ3 outside the ligand binding site

    PubMed Central

    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

  2. Rational design of a protein that binds integrin αvβ3 outside the ligand binding site.

    PubMed

    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

  3. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

    PubMed

    Heusser, Stephanie A; Yoluk, Özge; Klement, Göran; Riederer, Erika A; Lindahl, Erik; Howard, Rebecca J

    2016-07-01

    The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate

  4. Multiple Sites of Type II Site Ligand (Luteolin and BMHPC) Regulation of Gene Expression in PC-3 Cells.

    PubMed

    Markaverich, Barry M; Vijjeswarapu, Mary

    2012-12-01

    Type II [(3)H]estradiol binding site ligands including luteolin (a naturally occurring bioflavonoid) and synthetic compounds such as 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)cyclohexanone (BMHPC) inhibit normal and malignant prostate cell (PC-3, LNCaP, DU-145) proliferation in vitro and in vivo. Type II sites represent a binding domain on histone H4 possibly involved in an epigenetic mechanism for controlling gene transcription. Treatment of PC-3 human prostate cancer cells with luteolin or BMHPC modulated the expression of a number of genes in the epidermal growth factor receptor signaling pathway (EGFRSP) and cell cycle pathway (CCP). Pronounced stimulation (400-2000% of control) of c-FOS and p21 RNA expression was observed, suggesting that these were primary sites of action. Both compounds also caused irreversible G2/M arrest (p<0.001). siRNA's for c-FOS or p21 reduced the RNA expression of their respective targets by 85-95%, with minimal effects on cell proliferation. Furthermore, neither siRNA alone (single knockdown), or in combination (double knockdown), blocked luteolin or BMHPC inhibition of PC-3 cell proliferation. Thus, although c-FOS and p21 are known to modulate the expression of genes in the ESGRSP (EGFR, SOS, GRB2, JNK1, MKK4, RasGAP) and CCP (CCNA2, CCNE2, CDC25A, CDKN1A, CDKN1B, p27, PLK1) involved in the regulation of cell proliferation by luteolin and BMHPC, the c-FOS and p21 siRNA knockdown studies reported here suggest that c-FOS and p21 may be secondary bystanders in the overall response to these ligands in the regulation of PC-3 cell proliferation. PMID:23675277

  5. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  6. Exploration of the ligand binding site of the human 5-HT4 receptor by site-directed mutagenesis and molecular modeling

    PubMed Central

    Mialet, Jeanne; Dahmoune, Yamina; Lezoualc'h, Frank; Berque-Bestel, Isabelle; Eftekhari, Pierre; Hoebeke, Johan; Sicsic, Sames; Langlois, Michel; Fischmeister, Rodolphe

    2000-01-01

    Among the five human 5-HT4 (h5-HT4) receptor isoforms, the h5-HT4(a) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT4 receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells.Ligand binding or competition studies with two h5-HT4 receptor agonists, serotonin and ML10302 and two h5-HT4 receptor antagonists, [3H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase.Ligand binding experiments revealed that [3H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT4(a) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [3H]-GR113808 and to serotonin.According to these results, we propose ligand-receptor complex models with serotonin and [3H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [3H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [3H]-GR113808. PMID:10821780

  7. Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact.

    PubMed

    Collins, Brian E; Blixt, Ola; DeSieno, Alexis R; Bovin, Nicolai; Marth, Jamey D; Paulson, James C

    2004-04-20

    CD22, a negative regulator of B cell signaling, is a member of the siglec family that binds to alpha2-6-linked sialic acids on glycoproteins. Previous reports demonstrated that binding of multivalent sialoside probes to CD22 is blocked, or "masked," by endogenous (cis) ligands, unless they are first destroyed by sialidase treatment. These results suggest that cis ligands on B cells make CD22 functionally unavailable for binding to ligands in trans. Through immunofluorescence microscopy, however, we observed that CD22 on resting B cells redistributes to the site of contact with other B or T lymphocytes. Redistribution is mediated by interaction with trans ligands on the opposing cell because it does not occur with ligand-deficient lymphocytes from ST6GalI-null mice. Surprisingly, CD45, proposed as both a cis and trans ligand of CD22, was not required for redistribution to sites of cell contact, given that redistribution of CD22 was independent of CD45 and was observed with lymphocytes from CD45-deficient mice. Furthermore, CD45 is not required for CD22 masking as similar levels of masking were observed in the WT and null mice. Comparison of the widely used sialoside-polyacrylamide probe with a sialoside-streptavidin probe revealed that the latter bound a subset of B cells without sialidase treatment, suggesting that cis ligands differentially impacted the binding of these two probes in trans. The combined results suggest that equilibrium binding to cis ligands does not preclude binding of CD22 to ligands in trans, and allows for its redistribution to sites of contact between lymphocytes. PMID:15079087

  8. A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

    PubMed Central

    Cardó-Vila, Marina; Zurita, Amado J.; Giordano, Ricardo J.; Sun, Jessica; Rangel, Roberto; Guzman-Rojas, Liliana; Anobom, Cristiane D.; Valente, Ana P.; Almeida, Fábio C. L.; Lahdenranta, Johanna; Kolonin, Mikhail G.; Arap, Wadih; Pasqualini, Renata

    2008-01-01

    Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs. PMID:18941632

  9. Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

    SciTech Connect

    Sidhu, A.; Kassis, S.; Kebabian, J.; Fishman, P.H.

    1986-10-21

    An iodinated compound, (/sup 125/I)-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol, has been recently reported to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained the ability to functionally couple to the HeLa adenylate cyclase. These results suggest that the D-1 dopamine receptor contains NEM-sensitive sulfhydryl group(s) either at or near the vicinity of the ligand binding sites, which are critical for both receptor binding and function.

  10. Utilizing the Trispyrazolyl Borate Ligand for the Mimicking of O2-Activating Mononuclear Nonheme Iron Enzymes.

    PubMed

    Sallmann, Madleen; Limberg, Christian

    2015-10-20

    Mononuclear, O2-activating nonheme iron enzymes are a fascinating class of metalloproteines, capable of realizing the most different reactions, ranging from C-H activation, via O atom transfer to C-C bond cleavage, in the course of O2 activation. They can lead us the way to achieve similar reactions with comparable efficiency and selectivity in chemical laboratories, which would be highly desirable aiming at accessing value-added products or to achieve degradation of unwanted compounds. Hence, these enyzmes motivate attempts to construct artificial low-molecular weight analogues, mimicking structural or functional characteristics. Such models can, for instance, provide insights about which of the features inherent to an active site are essential and guarantee the enzyme function, and from this kind of information the minimal requirements for a biomimetic or bioinspired complex that may be applied in catalysis can be derived. On the other hand, they can contribute to an understanding of the enzyme functioning. In order to create such replicates, it is important to faithfully mimic the surroundings of the iron centers in their active sites. Most of them feature two histidine residues and one carboxylate donor, while a few exhibit a deceptively simple (His)3Fe active site. For the simulation of these, the trispyrazolyl borate ligand (Tp) particularly offers itself, as the facial arrangement of three pyrazole donors is reminiscent of the three histidine-derived imidazole donors. The focus of this Account will be on bioinorganic/biomimetic research from our laboratory utilizing Tp ligands to develop molecular models for (i) two representatives of the (His)3Fe-enzyme family, namely, the cysteine dioxygenase (CDO) and acetyl acetone dioxygenase (Dke1), (ii) a related but less well-explored variant of the CDO-the 2-aminoethanethiol dioxygenase-as well as (iii) the 2-His-1-carboxylate representative 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). The CDO catalyzes the

  11. Pharmacophore modeling using Site-Identification by Ligand Competitive Saturation (SILCS) with multiple probe molecules

    PubMed Central

    Yu, Wenbo; Lakkaraju, Sirish Kaushik; Raman, E. Prabhu; Fang, Lei; MacKerell, Alexander D.

    2015-01-01

    Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues as SILCS naturally takes both protein flexibility and desolvation effects into account by using full MD simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, re-ranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design. PMID:25622696

  12. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand.

    PubMed

    Sun, Qi; Phan, Jason; Friberg, Anders R; Camper, DeMarco V; Olejniczak, Edward T; Fesik, Stephen W

    2014-09-01

    K-Ras is a well-validated cancer target but is considered to be "undruggable" due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets. PMID:25087006

  13. Protonation Equilibria of Biologically Active Ligands in Mixed Aqueous Organic Solvents

    PubMed Central

    El-Sherif, Ahmed A.; Shoukry, Mohamed M.; Abd Elkarim, Abeer T.; Barakat, Mohammad H.

    2014-01-01

    The review is mainly concerned with the protonation equilibria of biologically active ligands like amino acids, peptides, DNA constituents, and amino acid esters in nonaqueous media. Equilibrium concentrations of proton-ligand formation as a function of pH were investigated. Also, thermodynamics associated with protonation equilibria were also discussed. PMID:25197267

  14. Aminotroponiminates as tunable, redox-active ligands: reversible single electron transfer and reductive dimerisation.

    PubMed

    Lichtenberg, C; Krummenacher, I

    2016-08-21

    Aminotroponiminates (atis) are shown to be redox-active ligands. Under strongly reducing conditions, the result of electron transfer can be controlled by the choice of the metal bound to the ati ligand. Either reversible electron transfer or a reductively induced dimerisation is observed. The latter reaction is (regio- and diastereo-) selective and chemically reversible. PMID:27452905

  15. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    PubMed Central

    Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  16. eMatchSite: Sequence Order-Independent Structure Alignments of Ligand Binding Pockets in Protein Models

    PubMed Central

    Brylinski, Michal

    2014-01-01

    Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4–9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite. PMID

  17. Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

    PubMed Central

    de Nobel, H; Lipke, P N; Kurjan, J

    1996-01-01

    The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin. Images PMID:8741846

  18. Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design.

    PubMed

    Faller, Christina E; Raman, E Prabhu; MacKerell, Alexander D; Guvench, Olgun

    2015-01-01

    Fragment-based drug design (FBDD) involves screening low molecular weight molecules ("fragments") that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind nonoverlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy.The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is "soaked" in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called "FragMaps" can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine "Grid Free Energies (GFEs)," which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

  19. Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

    PubMed Central

    Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

    2015-01-01

    Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy. The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is “soaked” in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called “FragMaps” can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine “Grid Free Energies (GFEs),” which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

  20. Selective Electrocatalytic Activity of Ligand Stabilized Copper Oxide Nanoparticles

    SciTech Connect

    Kauffman, Douglas R; Ohodnicki, Paul R; Kail, Brian W; Matranga, Christopher

    2011-01-01

    Ligand stabilization can influence the surface chemistry of Cu oxide nanoparticles (NPs) and provide unique product distributions for electrocatalytic methanol (MeOH) oxidation and CO{sub 2} reduction reactions. Oleic acid (OA) stabilized Cu{sub 2}O and CuO NPs promote the MeOH oxidation reaction with 88% and 99.97% selective HCOH formation, respectively. Alternatively, CO{sub 2} is the only reaction product detected for bulk Cu oxides and Cu oxide NPs with no ligands or weakly interacting ligands. We also demonstrate that OA stabilized Cu oxide NPs can reduce CO{sub 2} into CO with a {approx}1.7-fold increase in CO/H{sub 2} production ratios compared to bulk Cu oxides. The OA stabilized Cu oxide NPs also show 7.6 and 9.1-fold increases in CO/H{sub 2} production ratios compared to weakly stabilized and non-stabilized Cu oxide NPs, respectively. Our data illustrates that the presence and type of surface ligand can substantially influence the catalytic product selectivity of Cu oxide NPs.

  1. A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.

    PubMed

    He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E

    2011-03-01

    Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family. PMID:21226535

  2. Elucidation of distinct ligand binding sites for cytochrome P450 3A4.

    PubMed

    Hosea, N A; Miller, G P; Guengerich, F P

    2000-05-23

    Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M(r) 521) fits within P450 3A4. YPFP-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences in the K(i) values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to

  3. Two- and Three-Electron Oxidation of Single-Site Vanadium Centers at Surfaces by Ligand Design.

    PubMed

    Skomski, Daniel; Tempas, Christopher D; Cook, Brian J; Polezhaev, Alexander V; Smith, Kevin A; Caulton, Kenneth G; Tait, Steven L

    2015-06-24

    Rational, systematic tuning of single-site metal centers on surfaces offers a new approach to increase selectivity in heterogeneous catalysis reactions. Although such metal centers of uniform oxidation states have been achieved, the ability to control their oxidation states through the use of carefully designed ligands had not been shown. To this end, tetrazine ligands functionalized by two pyridinyl or pyrimidinyl substituents were deposited, along with vanadium metal, on the Au(100) surface. The greater oxidizing power of the bis-pyrimidinyltetrazine facilitates the on-surface redox formation of V(3+), compared to V(2+) when paired with the bis-pyridinyltetrazine, as determined by X-ray photoelectron spectroscopy. This demonstrates the ability to control metal oxidation states in surface coordination architectures by altering the redox properties of organic ligands. The metal-ligand complexes take the form of one-dimensional polymeric chains, resolved by scanning tunneling microscopy. The chain structures in the first layer are very uniform and are based on the same quasi-square-planar coordination geometry around single-site V with either ligand. Formation of a different, dimer structure is observed in the early stages of the second layer formation. These systems offer new opportunities in controlling the oxidation state of single-site transition metal atoms at a surface for new advances in heterogeneous catalysts. PMID:26029790

  4. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

    PubMed

    Zhang, Wei-Jie; Luo, Xiao; Liu, Ya-Li; Shao, Xiao-Xia; Wade, John D; Bathgate, Ross A D; Guo, Zhan-Yun

    2012-08-01

    Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various

  5. Multidentate terephthalamidate and hydroxypyridonate ligands: towards new orally active chelators.

    PubMed

    Abergel, Rebecca J; Raymond, Kenneth N

    2011-01-01

    The limitations of current therapies for the treatment of iron overload or radioisotope contamination have stimulated efforts to develop new orally bioavailable iron and actinide chelators. Siderophore-inspired tetradentate, hexadentate and octadentate terephthalamidate and hydroxypyridonate ligands were evaluated in vivo as selective and efficacious iron or actinide chelating agents, with several metal loading and ligand assessment procedures, using (59)Fe, (238)Pu, and (241)Am as radioactive tracers. The compounds presented in this study were compared to commercially available therapeutic sequestering agents [deferoxamine (DFO) for iron and diethylenetriaminepentaacetic acid (DPTA) for actinides] and are unrivaled in terms of affinity, selectivity and decorporation efficacy, which attests to the fact that high metal affinity may overcome the low bioavailability properties commonly associated to multidenticity. PMID:21599440

  6. MULTIDENTATE TEREPHTHALAMIDATE AND HYDROXYPYRIDONATE LIGANDS: TOWARDS NEW ORALLY ACTIVE CHELATORS

    SciTech Connect

    Abergel, Rebecca J.; Raymond, Kenneth N.

    2011-07-13

    The limitations of current therapies for the treatment of iron overload or radioisotope contamination have stimulated efforts to develop new orally bioavailable iron and actinide chelators. Siderophore-inspired tetradentate, hexadentate and octadentate terephthalamidate and hydroxypyridonate ligands were evaluated in vivo as selective and efficacious iron or actinide chelating agents, with several metal loading and ligand assessment procedures, using {sup 59}Fe, {sup 238}Pu, and {sup 241}Am as radioactive tracers. The compounds presented in this study were compared to commercially available therapeutic sequestering agents [deferoxamine (DFO) for iron and diethylenetriaminepentaacetic acid (DPTA) for actinides] and are unrivaled in terms of affinity, selectivity and decorporation efficacy, which attests to the fact that high metal affinity may overcome the low bioavailability properties commonly associated to multidenticity.

  7. Molecular models of site-isolated cobalt, rhodium, and iridium catalysts supported on zeolites: Ligand bond dissociation energies

    DOE PAGESBeta

    Chen, Mingyang; Serna, Pedro; Lu, Jing; Gates, Bruce C.; Dixon, David A.

    2015-09-28

    The chemistry of zeolite-supported site-isolated cobalt, rhodium, and iridium complexes that are essentially molecular was investigated with density functional theory (DFT) and the results compared with experimentally determined spectra characterizing rhodium and iridium species formed by the reactions of Rh(C2H4)2(acac) and Ir(C2H4)2(acac) (acac = acetylacetonate) with acidic zeolites such as dealuminated HY zeolite. The experimental results characterize ligand exchange reactions and catalytic reactions of adsorbed ligands, including olefin hydrogenation and dimerization. Two molecular models were used to characterize various binding sites of the metal complexes in the zeolites, and the agreement between experimental and calculated infrared frequencies and metal-ligand distancesmore » determined by extended X-ray absorption fine structure spectroscopy was generally very good. The calculated structures and energies indicate a metal-support-oxygen (M(I)-O) coordination number of two for most of the supported complexes and a value of three when the ligands include the radicals C2H5 or H. The results characterizing various isomers of the supported metal complexes incorporating hydrocarbon ligands indicate that some carbene and carbyne ligands could form. Ligand bond dissociation energies (LDEs) are reported to explain the observed reactivity trends. The experimental observations of a stronger M-CO bond than M-(C2H4) bond for both Ir and Rh match the calculated LDEs, which show that the single-ligand LDEs of the mono and dual-ligand complexes for CO are similar to 12 and similar to 15 kcal/mol higher in energy (when the metal is Rh) and similar to 17 and similar to 20 kcal/mol higher (when the metal is Ir) than the single-ligand LDEs of the mono and dual ligand complexes for C2H4, respectively. The results provide a foundation for the prediction of the catalytic properties of numerous supported metal complexes, as summarized in detail here.« less

  8. Molecular models of site-isolated cobalt, rhodium, and iridium catalysts supported on zeolites: Ligand bond dissociation energies

    SciTech Connect

    Chen, Mingyang; Serna, Pedro; Lu, Jing; Gates, Bruce C.; Dixon, David A.

    2015-09-28

    The chemistry of zeolite-supported site-isolated cobalt, rhodium, and iridium complexes that are essentially molecular was investigated with density functional theory (DFT) and the results compared with experimentally determined spectra characterizing rhodium and iridium species formed by the reactions of Rh(C2H4)2(acac) and Ir(C2H4)2(acac) (acac = acetylacetonate) with acidic zeolites such as dealuminated HY zeolite. The experimental results characterize ligand exchange reactions and catalytic reactions of adsorbed ligands, including olefin hydrogenation and dimerization. Two molecular models were used to characterize various binding sites of the metal complexes in the zeolites, and the agreement between experimental and calculated infrared frequencies and metal-ligand distances determined by extended X-ray absorption fine structure spectroscopy was generally very good. The calculated structures and energies indicate a metal-support-oxygen (M(I)-O) coordination number of two for most of the supported complexes and a value of three when the ligands include the radicals C2H5 or H. The results characterizing various isomers of the supported metal complexes incorporating hydrocarbon ligands indicate that some carbene and carbyne ligands could form. Ligand bond dissociation energies (LDEs) are reported to explain the observed reactivity trends. The experimental observations of a stronger M-CO bond than M-(C2H4) bond for both Ir and Rh match the calculated LDEs, which show that the single-ligand LDEs of the mono and dual-ligand complexes for CO are similar to 12 and similar to 15 kcal/mol higher in energy (when the metal is Rh) and similar to 17 and similar to 20 kcal/mol higher (when the metal is Ir) than the single-ligand LDEs of the mono and dual ligand complexes for C2H4, respectively. The results provide a foundation for the prediction

  9. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  10. Stability and structure of activated macrocyles. Ligand with biological applications

    SciTech Connect

    Motekaitis, R.J.; Martell, A.E.

    1996-06-19

    Single p-toluic acid pendant groups were attached to 1,4,7,10,13-pentaazacyclopentadecane (15aneN5) and 1,4,8,11-tetraazacyclotetradecane (cyclam) to prepare bifunctional reagents for radiolabeling monoclonal antibodies with {sup 64, 67}Cu. The ligands are 1,4,7,10,13-pentaazacyclopentadecane-1-({alpha}-1,4-toluic acid) (PCBA) and 1,4,8,11-tetraazacyclotetradecane-1-({alpha}-1,4-toluic acid) (CPTA). For the parent macrocycles and their pendant arm derivatives, the 1:1 Cu{sup 2+} complexes dissociate only below pH 2. At pH 0.0 and 25 {degrees}C the CPTA-Cu complex has a half-life toward complete dissociation of 24 days. A new approach was developed for the estimation of the Cu{sup 2+} stability constant for the kinetically robust CPTA. All other formation constants were determined at 25.0 {degrees}C with batch spectrophotometric techniques. Potentiometric titrations were used to determine the protonation constants of the macrocyclic ligands as well as of the metal chelates. The protonation constants, stability constants, and pM`s are discussed in terms of both molecular mechanics calculations and the ligands` potential applicability as copper(II) radiopharmaceuticals.

  11. Ligand Lone-Pair Influence on Hydrocarbon C-H Activation. A Computational Perspective

    SciTech Connect

    Ess, Daniel H.; Gunnoe, T. Brent; Cundari, Thomas R.; Goddard, William A.; Periana, Roy A.

    2010-12-03

    Mid to late transition metal complexes that break hydrocarbon C-H bonds by transferring the hydrogen to a heteroatom ligand while forming a metal-alkyl bond offer a promising strategy for C-H activation. Here we report a density functional (B3LYP, M06, and X3LYP) analysis of cis-(acac)2MX and TpM(L)X (M = Ir, Ru, Os, and Rh; acac = acetylacetonate, Tp = tris(pyrazolyl)borate; X = CH3, OH, OMe, NH2, and NMe2) systems for methane C-H bond activation reaction kinetics and thermodynamics. We address the importance of whether a ligand lone pair provides an intrinsic kinetic advantage through possible electronic dπ-pπ repulsions for M-OR and M-NR2 systems versus M-CH3 systems. This involves understanding the energetic impact of the X ligand group on ligand loss, C-H bond coordination, and C-H bond cleavage steps as well as understanding how the nucleophilicity of the ligand X group, the electrophilicity of the transition metal center, and cis-ligand stabilization effect influence each of these steps. We also explore how spectator ligands and second- versus third-row transition metal centers impact the energetics of each of these C-H activation steps.

  12. Recognition of Mannosylated Ligands and Influenza A Virus by Human Surfactant Protein D: Contributions of an Extended Site and Residue 343

    SciTech Connect

    Crouch, E.; Hartshorn, K; Horlacher, T; McDonald, B; Smith, K; Cafarella, T; Seaton, B; Seeberger, P; Head, J

    2009-01-01

    Surfactant protein D (SP-D) plays important roles in antiviral host defense. Although SP-D shows a preference for glucose/maltose, the protein also recognizes d-mannose and a variety of mannose-rich microbial ligands. This latter preference prompted an examination of the mechanisms of mannose recognition, particularly as they relate to high-mannose viral glycans. Trimeric neck plus carbohydrate recognition domains from human SP-D (hNCRD) preferred ?1-2-linked dimannose (DM) over the branched trimannose (TM) core, ?1-3 or ?1-6 DM, or d-mannose. Previous studies have shown residues flanking the carbohydrate binding site can fine-tune ligand recognition. A mutant with valine at 343 (R343V) showed enhanced binding to mannan relative to wild type and R343A. No alteration in affinity was observed for d-mannose or for ?1-3- or ?1-6-linked DM; however, substantially increased affinity was observed for ?1-2 DM. Both proteins showed efficient recognition of linear and branched subdomains of high-mannose glycans on carbohydrate microarrays, and R343V showed increased binding to a subset of the oligosaccharides. Crystallographic analysis of an R343V complex with 1,2-DM showed a novel mode of binding. The disaccharide is bound to calcium by the reducing sugar ring, and a stabilizing H-bond is formed between the 2-OH of the nonreducing sugar ring and Arg349. Although hNCRDs show negligible binding to influenza A virus (IAV), R343V showed markedly enhanced viral neutralizing activity. Hydrophobic substitutions for Arg343 selectively blocked binding of a monoclonal antibody (Hyb 246-05) that inhibits IAV binding activity. Our findings demonstrate an extended ligand binding site for mannosylated ligands and the significant contribution of the 343 side chain to specific recognition of multivalent microbial ligands, including high-mannose viral glycans.

  13. The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site.

    PubMed

    Knospe, Melanie; Müller, Christa E; Rosa, Patrizia; Abdelrahman, Aliaa; von Kügelgen, Ivar; Thimm, Dominik; Schiedel, Anke C

    2013-09-01

    The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors. PMID:23413038

  14. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

    PubMed Central

    Pittman, R C; Carew, T E; Glass, C K; Green, S R; Taylor, C A; Attie, A D

    1983-01-01

    We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples. PMID:6882394

  15. Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites

    NASA Astrophysics Data System (ADS)

    Vukovic, Sinisa; Brennan, Paul E.; Huggins, David J.

    2016-09-01

    The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

  16. Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites.

    PubMed

    Vukovic, Sinisa; Brennan, Paul E; Huggins, David J

    2016-09-01

    The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process. PMID:27367338

  17. Structure-activity relationship study of non-steroidal NPC1L1 ligands identified through cell-based assay using pharmacological chaperone effect as a readout.

    PubMed

    Karaki, Fumika; Ohgane, Kenji; Fukuda, Hiromitsu; Nakamura, Masahiko; Dodo, Kosuke; Hashimoto, Yuichi

    2014-07-15

    Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure-activity relationships of their derivatives as NPC1L1 ligands. PMID:24906511

  18. Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17*

    PubMed Central

    Habets, Roger A. J.; Groot, Arjan J.; Yahyanejad, Sanaz; Tiyanont, Kittichoat; Blacklow, Stephen C.; Vooijs, Marc

    2015-01-01

    Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and γ-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs. PMID:25918160

  19. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    PubMed

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  20. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

    PubMed Central

    Ukhanov, K.; Bobkov, Y.; Corey, E.A.; Ache, B.W.

    2014-01-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  1. Water oxidation chemistry of a synthetic dinuclear ruthenium complex containing redox-active quinone ligands.

    PubMed

    Isobe, Hiroshi; Tanaka, Koji; Shen, Jian-Ren; Yamaguchi, Kizashi

    2014-04-21

    We investigated theoretically the catalytic mechanism of electrochemical water oxidation in aqueous solution by a dinuclear ruthenium complex containing redox-active quinone ligands, [Ru2(X)(Y)(3,6-tBu2Q)2(btpyan)](m+) [X, Y = H2O, OH, O, O2; 3,6-tBu2Q = 3,6-di-tert-butyl-1,2-benzoquinone; btpyan =1,8-bis(2,2':6',2″-terpyrid-4'-yl)anthracene] (m = 2, 3, 4) (1). The reaction involves a series of electron and proton transfers to achieve redox leveling, with intervening chemical transformations in a mesh scheme, and the entire molecular structure and motion of the catalyst 1 work together to drive the catalytic cycle for water oxidation. Two substrate water molecules can bind to 1 with simultaneous loss of one or two proton(s), which allows pH-dependent variability in the proportion of substrate-bound structures and following pathways for oxidative activation of the aqua/hydroxo ligands at low thermodynamic and kinetic costs. The resulting bis-oxo intermediates then undergo endothermic O-O radical coupling between two Ru(III)-O(•) units in an anti-coplanar conformation leading to bridged μ-peroxo or μ-superoxo intermediates. The μ-superoxo species can liberate oxygen with the necessity for the preceding binding of a water molecule, which is possible only after four-electron oxidation is completed. The magnitude of catalytic current would be limited by the inherent sluggishness of the hinge-like bending motion of the bridged μ-superoxo complex that opens up the compact, hydrophobic active site of the catalyst and thereby allows water entry under dynamic conditions. On the basis of a newly proposed mechanism, we rationalize the experimentally observed behavior of electrode kinetics with respect to potential and discuss what causes a high overpotential for water oxidation by 1. PMID:24694023

  2. Implication of crystal water molecules in inhibitor binding at ALR2 active site.

    PubMed

    Hymavati; Kumar, Vivek; Sobhia, M Elizabeth

    2012-01-01

    Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. PMID:22649481

  3. Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

    PubMed

    Liantonio, Antonella; Picollo, Alessandra; Carbonara, Giuseppe; Fracchiolla, Giuseppe; Tortorella, Paolo; Loiodice, Fulvio; Laghezza, Antonio; Babini, Elena; Zifarelli, Giovanni; Pusch, Michael; Camerino, Diana Conte

    2008-01-29

    ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels. PMID:18216243

  4. New avenues for ligand-mediated processes--expanding metal reactivity by the use of redox-active catechol, o-aminophenol and o-phenylenediamine ligands.

    PubMed

    Broere, Daniël L J; Plessius, Raoul; van der Vlugt, Jarl Ivar

    2015-10-01

    Redox-active ligands have evolved from being considered spectroscopic curiosities - creating ambiguity about formal oxidation states in metal complexes - to versatile and useful tools to expand on the reactivity of (transition) metals or to even go beyond what is generally perceived possible. This review focusses on metal complexes containing either catechol, o-aminophenol or o-phenylenediamine type ligands. These ligands have opened up a new area of chemistry for metals across the periodic table. The portfolio of ligand-based reactivity invoked by these redox-active entities will be discussed. This ranges from facilitating oxidative additions upon d(0) metals or cross coupling reactions with cobalt(iii) without metal oxidation state changes - by functioning as an electron reservoir - to intramolecular ligand-to-substrate single-electron transfer to create a reactive substrate-centered radical on a Pd(ii) platform. Although the current state-of-art research primarily consists of stoichiometric and exploratory reactions, several notable reports of catalysis facilitated by the redox-activity of the ligand will also be discussed. In conclusion, redox-active ligands containing catechol, o-aminophenol or o-phenylenediamine moieties show great potential to be exploited as reversible electron reservoirs, donating or accepting electrons to activate substrates and metal centers and to enable new reactivity with both early and late transition as well as main group metals. PMID:26148803

  5. Peripheral site ligand conjugation to a non-quaternary oxime enhances reactivation of nerve agent-inhibited human acetylcholinesterase.

    PubMed

    de Koning, Martijn C; van Grol, Marco; Noort, Daan

    2011-09-25

    Commonly employed pyridinium-oxime (charged) reactivators of nerve agent inhibited acetylcholinesterase (AChE) do not readily pass the blood brain barrier (BBB) because of the presence of charge(s). Conversely, non-ionic oxime reactivators often suffer from a lack of reactivating potency due to a low affinity for the active site of AChE. It was therefore hypothesized that an extra contribution in affinity may be achieved by covalently connecting a peripheral site ligand (PSL) to a non-ionic reactivator, which may result in a higher reactivation potency of the total construct. This validity of this approach, which proved successful for charged pyridinium oximes in earlier work, is now further exemplified with the covalent linkage of a neutral PSL via a spacer to a non-ionic and otherwise almost non-reactivating α-ketoaldoxime. It is demonstrated that the linkage of the PSL resulted in a remarkable increase in reactivation potency of the hybrid compounds. Although the molecules reported here are still inefficient reactivators compared to the current pyridinium oximes, the presented approach holds promise for the future design and synthesis of non-ionic oxime reactivators with improved BBB penetration and may be suited as well for non-oxime reactivators thus further widening the scope in the ongoing search for broad-spectrum reactivators. PMID:21504785

  6. Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling

    PubMed Central

    Liuzzi, Anna Rita; Kift-Morgan, Ann; Lopez-Anton, Melisa; Friberg, Ida M.; Zhang, Jingjing; Brook, Amy C.; Roberts, Gareth W.; Donovan, Kieron L.; Colmont, Chantal S.; Toleman, Mark A.; Bowen, Timothy; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Fraser, Donald J.

    2016-01-01

    The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2+ γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response. PMID:27527598

  7. (3H) 5,7-dichlorokynurenic acid, a high affinity ligand for the NMDA receptor glycine regulatory site

    SciTech Connect

    Hurt, S.D.; Baron, B.M. )

    1991-01-01

    The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. The authors have prepared (3H) 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield (3H) 5,7-DCKA with a specific activity of 17.6 Ci/mmol. (3H) 5,7-DCKA bound to rat brain synaptosomes with a Kd of 69 {plus minus} 23 nM and Bmax = 14.5 {plus minus} 3.2 pmoles/mg protein. Binding was 65-70% specific at 10 nM (3H) 5,7-DCKA. This ligand is thus more selective and has higher affinity than (3H) glycine, in addition to being an antagonist.

  8. Ligand-dependent Enhancer Activation Regulated by Topoisomerase-I Activity

    PubMed Central

    Puc, Janusz; Kozbial, Piotr; Li, Wenbo; Tan, Yuliang; Liu, Zhijie; Suter, Tom; Ohgi, Kenneth A.; Zhang, Jie; Aggarwal, Aneel K.; Rosenfeld, Michael G.

    2014-01-01

    SUMMARY The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation, and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs. PMID:25619691

  9. A fluorescent reporter detects details of aromatic ligand interference in drug-binding sites of human serum albumin.

    PubMed

    Dobretsov, Gennady; Smolina, Natalia; Syrejshchikova, Tatiana; Brilliantova, Varvara; Uzbekov, Marat

    2016-09-01

    Human serum albumin (HSA) transports many ligands including small aromatic molecules: metabolites, drugs etc. Phenylbutazone is an anti-inflammatory drug, which binds to the drug-binding site I of HSA. Its interaction with this site has been studied using a fluorescent dye, CAPIDAN, whose fluorescence in serum originates from HSA and is sensitive to the changes in HSA site I in some diseases. Its fluorescence in HSA solutions is strongly suppressed by phenylbutazone. This phenomenon seems to be a basic sign of a simple drug-dye competition. However, a more detailed study of the time-resolved fluorescence decay of CAPIDAN has shown that phenylbutazone lowers fluorescence without changing the total amount of bound dye. In brief, the HSA-bound dye forms three populations due to three types of environment at the binding sites. The first two populations probably have a rather strong Coulomb interaction with the positive charge of residues Arginine 218 or Arginine 222 in site I and are responsible for approximately 90% of the total fluorescence. Phenylbutazone blocks this interaction and therefore lowers this fluorescence. At the same time the binding of the third population increases considerably in the presence of phenylbutazone, and, as a result, the actual number of bound dye molecules remains almost unchanged despite the ligand competition. So, time resolved fluorescence of the reporter allows to observe details of interactions and interference of aromatic ligands in drug binding site I of HSA both in isolated HSA and in serum. PMID:27318089

  10. Selective Targeting of the TPX2 Site of Importin-α Using Fragment-Based Ligand Design.

    PubMed

    Holvey, Rhian S; Valkov, Eugene; Neal, David; Stewart, Murray; Abell, Chris

    2015-07-01

    Protein-protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2-importin-α interaction. Importin-α is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites--major and minor-to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment-based approaches were used to identify small molecules that bind importin-α, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure-guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein-protein interaction. PMID:25899172

  11. Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme–Ligand Promiscuity Revealed at the Atomistic Level

    PubMed Central

    2015-01-01

    Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of l- to d-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered molecular dynamics (MD), docking, and experimental studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-associated changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-associated phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly associated with each of the GRY53/7HC–ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC–ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-associated GR changes are consistent with the experimentally observed quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and

  12. Inhibition of ligand-independent constitutive activation of the Met oncogenic receptor by the engineered chemically-modified antibody DN30.

    PubMed

    Vigna, Elisa; Chiriaco, Cristina; Cignetto, Simona; Fontani, Lara; Basilico, Cristina; Petronzelli, Fiorella; Comoglio, Paolo M

    2015-11-01

    An awesome number of experimental and clinical evidences indicate that constitutive activation of the Met oncogenic receptor plays a critical role in the progression of cancer toward metastasis and/or resistance to targeted therapies. While mutations are rare, the common mechanism of Met activation is overexpression, either by gene amplification ('addiction') or transcriptional activation ('expedience'). In the first instance ligand-independent kinase activation plays a major role in sustaining the transformed phenotype. Anti-Met antibodies directed against the receptor binding site behave essentially as ligand (Hepatocyte Growth Factor, HGF) antagonists and are ineffective to counteract ligand-independent activation. The monovalent chimeric MvDN30 antibody fragment, PEGylated to extend its half-life, binds the fourth IPT domain and induces 'shedding' of the Met extracellular domain, dramatically reducing both the number of receptors on the surface and their phosphorylation. Downstream signaling is thus inhibited, both in the absence or in the presence of the ligand. In vitro, MvDN30 is a strong inhibitor not only of ligand-dependent invasive growth, sustained by both paracrine and autocrine HGF, but notably, also of ligand-independent growth of 'Met-addicted' cells. In immunocompromised mice, lacking expression of Hepatocyte Growth Factor cross-reacting with the human receptor - thus providing, by definition, a model of 'ligand-independent' Met activation - PEGylated MvDN30 impairs growth of Met 'addicted' human gastric carcinoma cells. In a Met-amplified patient-derived colo-rectal tumor (xenopatient) MvDN30-PEG overcomes the resistance to EGFR targeted therapy (Cetuximab). The PEGylated MvDN30 is thus a strong candidate for targeting tumors sustained by ligand-independent Met oncogenic activation. PMID:26119717

  13. Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site.

    PubMed

    Pujadas, G; Palau, J

    2001-08-01

    Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center. PMID:11468361

  14. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection

    PubMed Central

    Deng, Weiwen; Gowen, Benjamin G.; Zhang, Li; Wang, Lin; Lau, Stephanie; Iannello, Alexandre; Xu, Jianfeng; Rovis, Tihana L.; Xiong, Na; Raulet, David H.

    2016-01-01

    Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, shedding of MULT1, a high affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are inhibitory, and suggest a new approach for cancer immunotherapy. PMID:25745066

  15. Activation of isocyanate ligands in Ru25+ complexes

    NASA Astrophysics Data System (ADS)

    Barral, M. Carmen; Herrero, Santiago; Jiménez-Aparicio, Reyes; Priego, José L.; Torres, M. Rosario; Urbanos, Francisco A.

    2008-11-01

    The reaction of [Ru 2(O 2CMe)(DPhF) 3(H 2O)]BF 4 · 0.5CH 2Cl 2 (DphF dbnd N, N'-diphenylformidinate) with sodium cyanate leads to the substitution of the H 2O ligand giving Ru 2(NCO)(O 2CMe)(DPhF) 3 ( 1). In contrast, in the similar reaction of Ru 2Cl 2(DPhF) 3 with NaOCN one of the cyanate groups undergoes the addition of a MeOH molecule leading to the carbamate complex Ru 2(NCO)(NH(O)COMe)(DPhF) 3 ( 2). The spectroscopic properties of 1 and 2 are studied. Both complexes are paramagnetic showing the presence of three unpaired electrons with an important zero-field splitting and a small intermolecular antiferromagnetic interaction. The crystal structures of 1 · 3CHCl 3 and 2 · C 7H 8 · 0.5MeOH are also reported. Compound 2 represents the first example of a ruthenium paddlewheel compound with a carbamate ligand.

  16. Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus;#8197;1

    SciTech Connect

    Wielens, Jerome; Headey, Stephen J.; Deadman, John J.; Rhodes, David I.; Parker, Michael W.; Chalmers, David K.; Scanlon, Martin J.

    2011-08-17

    Fragment-based screening has been used to identify a novel ligand binding site on HIV-1 integrase. Crystal structures of fragments bound at this site (shown) have been used to design elaborated second-generation compounds that bind with higher affinity and good ligand efficiency.

  17. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. PMID:25148941

  18. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  19. Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand derived from cinnamaldehyde.

    PubMed

    Shreaz, Sheikh; Shiekh, Rayees A; Raja, Vaseem; Wani, Waseem A; Behbehani, Jawad M

    2016-03-01

    In this study, we have used aldehyde function of cinnamaldehyde to synthesize N, N'-Bis (cinnamaldehyde) ethylenediimine [C20H20N2] and Co(II) complex of the type [Co(C40H40N4)Cl2]. The structures of the synthesized compounds were determined on the basis of physiochemical analysis and spectroscopic data ((1)H NMR, FTIR, UV-visible and mass spectra) along with molar conductivity measurements. Anticandidal activity of cinnamaldehyde its ligand [L] and Co(II) complex was investigated by determining MIC80, time-kill kinetics, disc diffusion assay and ergosterol extraction and estimation assay. Ligand [L] and Co(II) complex are found to be 4.55 and 21.0 folds more efficient than cinnamaldehyde in a liquid medium. MIC80 of Co(II) complex correlated well with ergosterol inhibition suggesting ergosterol biosynthesis to be the primary site of action. In comparison to fluconazole, the test compounds showed limited toxicity against H9c2 rat cardiac myoblasts. In confocal microscopy propidium iodide (PI) penetrates the yeast cells when treated with MIC of metal complex, indicating a disruption of cell membrane that results in imbibition of dye. TEM analysis of metal complex treated cells exhibited notable alterations or damage to the cell membrane and the cell wall. The structural disorganization within the cell cytoplasm was noted. It was concluded that fungicidal activity of Co(II) complex originated from loss of membrane integrity and a decrease in ergosterol content is only one consequence of this. PMID:26806515

  20. Synthesis, characterization and antibacterial activity of new Ln(III) complexes with an unsymmetrical schiff base ligand

    NASA Astrophysics Data System (ADS)

    Caifeng, Bi; Liangliang, Yan; Yuhua, Fan; Xia, Zhang; Aidong, Wang

    2006-07-01

    A new unsymmetrical Schiff base ligand (H2LLi) was synthesized using L-lysine, salicyladehyde and 2-hydroxy-1-naphthaldehyde. Three solid metal complexes of this ligand [Ln(H2L)(NO3)] NO3·2H2O (Ln=La, Sm, Ho) have been prepared and characterized by elemental analyses, IR spectra, UV spectra, TG-DTG and molar conductance. The antibacterial activities of the ligand and its complexes are also studied. The antibacterial experiments indicate that the ligand and its complexes possess antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis and that the complexes have higher activity than those of the ligand.

  1. Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation.

    PubMed

    Liu, Yuan-Tao; Song, Lifang; Templeton, Douglas M

    2007-04-01

    Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts. PMID:17226785

  2. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    PubMed

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  3. Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels.

    PubMed

    Basak, Sandip; Chatterjee, Soumili; Chakrapani, Sudha

    2016-01-01

    Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics. PMID:27403967

  4. The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand binding sites in a single aptamer.

    PubMed

    Trausch, Jeremiah J; Ceres, Pablo; Reyes, Francis E; Batey, Robert T

    2011-10-12

    Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role. PMID:21906956

  5. A surface site as polydentate ligand of a metal complex: Density functional studies of rhenium subcarbonyls supported on magnesium oxide

    SciTech Connect

    Hu, A.; Neyman, K.M.; Staufer, M.; Belling, T.; Gates, B.C.; Roesch, N.

    1999-05-12

    Notwithstanding the importance of supported organometallic species as industrial catalysts, most are nonuniform mixtures, with only a few being well-characterized at the atomic level. Rhenium subcarbonyls on MgO, in contrast, consist of nearly uniform surface species and are among the best-studied organometallic complexes on oxides. EXAFS and infrared spectra showed that decomposition of the precursors [HRe(CO){sub 5}], [H{sub 3}Re{sub 3}(CO){sub 12}], and [Re{sub 2}(CO){sub 10}] on MgO powder results in fragments, assigned as Re(CO){sub 3}{sup n+}, coordinated to surface ligands. The concept of a surface site as a polydentate ligand evokes the remarkable circumstance in which the adsorbate-substrate bonds are as strong as metal-ligand bonds in common transition metal complexes, as shown by the present investigation.

  6. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  7. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  8. Copper-Sulfur Complexes Supported by N-Donor Ligands: Towards Models of the Cu(Z) Site in Nitrous Oxide Reductase.

    PubMed

    York, John T; Bar-Nahum, Itsik; Tolman, William B

    2008-03-01

    The distinctive structure of the [(his)(7)Cu(4)(μ-S)](n+) cluster in the "Cu(Z)" active site of nitrous oxide reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper-sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper-sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper-sulfur bonding interactions in the complexes. PMID:19262681

  9. Quantitative conformationally sampled pharmacophore for delta opioid ligands: reevaluation of hydrophobic moieties essential for biological activity.

    PubMed

    Bernard, Denzil; Coop, Andrew; MacKerell, Alexander D

    2007-04-19

    Recent studies have indicated several therapeutic applications for delta opioid agonists and antagonists. To exploit the therapeutic potential of delta opioids developing a structural basis for the activity of ligands at the delta opioid receptor is essential. The conformationally sampled pharmacophore (CSP) method (Bernard et al. J. Am. Chem. Soc. 2003, 125, 3103-3107) is extended here to obtain quantitative models of delta opioid ligand efficacy and affinity. Quantification is performed via overlap integrals of the conformational space sampled by ligands with respect to a reference compound. Iterative refinement of the CSP model identified hydrophobic groups other than the traditional phenylalanine residues as important for efficacy and affinity in DSLET and ICI 174 864. The obtained models for a structurally diverse set of peptidic and nonpeptidic delta opioid ligands offer good predictions with R2 values>0.9, and the predicted efficacy for a set of test compounds was consistent with the experimental values. PMID:17367120

  10. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand

    PubMed Central

    Sun, Qi; Phan, Jason; Friberg, Anders R.; Camper, DeMarco V.; Olejniczak, Edward T.; Fesik, Stephen W.

    2015-01-01

    K-Ras is a well-validated cancer target but is considered to be “undruggable” due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets. PMID:25087006

  11. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  12. Ruthenium Dihydroxybipyridine Complexes are Tumor Activated Prodrugs Due to Low pH and Blue Light Induced Ligand Release

    PubMed Central

    Hufziger, Kyle T.; Thowfeik, Fathima Shazna; Charboneau, David J.; Nieto, Ismael; Dougherty, William G.; Kassel, W. Scott; Dudley, Timothy J.; Merino, Edward J.; Papish, Elizabeth T.; Paul, Jared J.

    2013-01-01

    Ruthenium drugs are potent anti-cancer agents, but inducing drug selectivity and enhancing their modest activity remain challenging. Slow Ru ligand loss limits the formation of free sites and subsequent binding to DNA base pairs. Herein, we designed a ligand that rapidly dissociates upon irradiation at low pH. Activation at low pH can lead to cancer selectivity, since many cancer cells have higher metabolism (and thus lower pH) than non-cancerous cells. We have used the pH sensitive ligand, 6,6′-dihydroxy-2,2′-bipyridine (66′bpy(OH)2), to generate [Ru(bpy)2(66′(bpy(OH)2)]2+, which contains two acidic hydroxyl groups with pKa1 = 5.26 and pKa2 = 7.27. Irradiation when protonated leads to photo-dissociation of the 66′bpy(OH)2 ligand. An in-depth study of the structural and electronic properties of the complex was carried out using X-Ray crystallography, electrochemistry, UV/visible spectroscopy, and computational techniques. Notably, Ru-N bond lengths in the 66′bpy(OH)2 complex are longer (by ~0.3 Å) than in polypyridyl complexes that lack 6 and 6′ substitution. Thus, the longer bond length predisposes the complex for photo-dissociation and leads to the anti-cancer activity. When the complex is deprotonated, the 66′bpy(O−)2 ligand molecular orbitals mix heavily with the ruthenium orbitals, making new mixed metal-ligand orbitals that lead to a higher bond order. We investigated the anti-cancer activities of [Ru(bpy)2(66′(bpy(OH)2)]2+, [Ru(bpy)2(44′(bpy(OH)2)]2+, and [Ru(bpy)3]2+ (44′(bpy(OH)2 = 4,4′-dihydroxy-2,2′-bipyridine) in HeLa cells, which have a relatively low pH. It is found that [Ru(bpy)2(66′(bpy(OH)2)]2+ is more cytotoxic than the other ruthenium complexes studied. Thus, we have identified a pH sensitive ruthenium scaffold that can be exploited for photo-induced anti-cancer activity. PMID:24184694

  13. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    PubMed

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels. PMID:24830141

  14. Interaction of tryptamine and ergoline compounds with threonine 196 in the ligand binding site of the 5-hydroxytryptamine6 receptor.

    PubMed

    Boess, F G; Monsma, F J; Meyer, V; Zwingelstein, C; Sleight, A J

    1997-09-01

    We examined the ligand-binding site of the 5-hydroxytryptamine6 (5-HT6) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of trans-membrane region V are important for ligand binding in several bioamine receptors. In the 5-HT6 receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [3H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [3H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30 fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mainserin, changed very little or increased. This indicates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT6 receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT6 receptor. PMID:9284367

  15. Automatic generation of bioinformatics tools for predicting protein–ligand binding sites

    PubMed Central

    Banno, Masaki; Ueki, Kokoro; Saad, Gul; Shimizu, Kentaro

    2016-01-01

    Motivation: Predictive tools that model protein–ligand binding on demand are needed to promote ligand research in an innovative drug-design environment. However, it takes considerable time and effort to develop predictive tools that can be applied to individual ligands. An automated production pipeline that can rapidly and efficiently develop user-friendly protein–ligand binding predictive tools would be useful. Results: We developed a system for automatically generating protein–ligand binding predictions. Implementation of this system in a pipeline of Semantic Web technique-based web tools will allow users to specify a ligand and receive the tool within 0.5–1 day. We demonstrated high prediction accuracy for three machine learning algorithms and eight ligands. Availability and implementation: The source code and web application are freely available for download at http://utprot.net. They are implemented in Python and supported on Linux. Contact: shimizu@bi.a.u-tokyo.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26545824

  16. Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces.

    PubMed Central

    Lorant, D E; McEver, R P; McIntyre, T M; Moore, K L; Prescott, S M; Zimmerman, G A

    1995-01-01

    In acute inflammatory responses, selectins mediate initial rolling of neutrophils (PMNs) along the endothelial surface. This is followed by tight adhesion that requires activation-dependent up-regulation of CD11/CD18 integrins on PMNs. For emigration to occur, the initial bonds that are established at the endothelial surface must be disengaged. We show that activation of PMNs results in their detachment from P-selectin, a glycoprotein expressed at the surface of inflamed endothelium that mediates initial tethering of PMNs. Loosening of the bond occurs when PMNs are activated by platelet-activating factor, which is coexpressed with P-selectin, or by other signaling molecules. The time course of reduced adhesion to P-selectin, when compared to up-regulation of CD11/CD18 integrins, suggests that "bond trading" may occur as activated PMNs transmigrate in vivo. Activation of PMNs did not alter binding of fluid-phase P-selectin, indicating that the ligand(s) for P-selectin is not shed or internalized. Using microspheres coated with P-selectin, we found that ligands for P-selectin were randomly distributed over the surfaces of rounded, unactivated PMNs. An antibody against P-selectin glycoprotein ligand-1 (PSGL-1) completely inhibited binding of P-selectin-coated beads suggesting that P-selectin glycoprotein ligand-1 is the critical binding site in this assay. In contrast to the dispersed pattern on unactivated PMNs, the ligands for P-selectin were localized on the uropods of activated, polarized cells. Pretreating PMNs with cytochalasin D before activation prevented the change in cell shape, the redistribution of binding sites for P-selectin-coated beads, and the decrease in cellular adhesiveness for P-selectin. These experiments indicate that the distribution of ligands for P-selectin is influenced by cellular activation and by cytoskeletal interactions, and that redistribution of these ligands may influence adhesive interactions. Activation of PMNs may cause loosening

  17. A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

    PubMed Central

    McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

    2015-01-01

    Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

  18. Synthesis, characterization and biological activity of new mixed ligand complexes of Zn(II) naproxen with nitrogen based ligands.

    PubMed

    Abu Ali, Hijazi; Fares, Hadeel; Darawsheh, Mohanad; Rappocciolo, Emilia; Akkawi, Mutaz; Jaber, Suhair

    2015-01-01

    A series of novel Zn(II) complexes [Zn2(nap)4] (1), [Zn(nap)21,10-phen](2), [Zn(nap)22,9-dmphen] (3), [Zn(nap)2(2-ampy)2] (4), [Zn(nap)2(imid)2] (5), [Zn(nap)2(1,2-dmimid)2] (6) (nap = naproxen, 1,10-phen = 1,10-phenanthroline, 2,9-dmphen = 2,9-dimethyl-1,10-phenanthroline, 2-ampy = 2-aminopyridine, imid = imidazole, 1,2-dmimid = 1,2-dimethyl imidazole) were synthesized and characterized using IR, UV-Vis, (1)H NMR, (13)C{(1)H} NMR spectroscopy. The crystal structure of complex 3 was determined using single-crystal X-ray diffraction. In order to assess the effect of the metal ions on the anti-bacterial activity, complexes 1-6 have been screened in vitro, against (G(+)) bacteria (Staphylococcus aureus and Micrococcus luteus) and (G(-)) bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli) using the agar well diffusion method. Complex 2 was the only complex that showed antibacterial activity against P. aeruginosa, where the complexation of the parent ligand 1,10-phenathroline enhanced significantly the activity. All the complexes showed different activity against the different bacteria, and were compared with activity of the parent ligands. The complexes were tested also for their anti-malarial activity using two methods: a semi-quantitative micro-assay and a previously self-developed quantitative in-vitro method. Both were used to study the efficiency of these complexes in inhibiting the formation of the Malaria pigment. This is considered an important target of many known anti-malarial drugs such as Chloroquine and Amodaquine. Results showed that the efficiency of complex 3 in preventing the formation of β-hematin was 75%. The efficiency of Amodiaquine as a standard drug was reported to give 92.5. PMID:25462227

  19. Copper coordinated ligand thioether-S and NO2(-) oxidation: relevance to the CuM site of hydroxylases.

    PubMed

    Maji, Ram Chandra; Bhandari, Anirban; Singh, Ravindra; Roy, Suprakash; Chatterjee, Sudip K; Bowles, Faye L; Ghiassi, Kamran B; Maji, Milan; Olmstead, Marilyn M; Patra, Apurba K

    2015-10-28

    In order to gain insight into the coordination site and oxidative activity of the CuM site of hydroxylases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM), we have synthesized, characterized and studied the oxidation chemistry of copper complexes chelated by tridentate N2Sthioether, N2Osulfoxide or N2Osulfone donor sets. The ligands are those of N-2-methylthiophenyl-2'-pyridinecarboxamide (HL1), and the oxidized variants, N-2-methylsulfenatophenyl-2'-pyridinecarboxamide (HL1(SO)), and N-2-methylsulfinatophenyl-2'-pyridinecarboxamide (HL1(SO2)). Our studies afforded the complexes [(L1)Cu(II)(H2O)](ClO4)·H2O (1·H2O), {[(L1(SO))Cu(II)(CH3CN)](ClO4)}n (2), [(L1)Cu(II)(ONO)] (3), [(L1(SO))Cu(II)(ONO)]n (4), [(L1)Cu(II)(NO3)]n (5), [(L1(SO))Cu(II)(NO3)]n (6) and [(L1(SO2))Cu(II)(NO3)] (7). Complexes 1 and 3 were described in a previous publication (Inorg. Chem., 2013, 52, 11084). The X-ray crystal structures revealed either distorted octahedral (in 2, 4-6) or square-pyramidal (in 1, 3) coordination geometry around Cu(II) ions of the complexes. In the presence of H2O2, conversion of 1→2, 3-5→6 and 6→7 occurs quantitatively via oxidation of thioether-S and/or Cu(ii) coordinated NO2(-) ions. Thioether-S oxidation of L1 also occurs when [L1](-) is reacted with [Cu(I)(CH3CN)4](ClO4) in DMF under O2, albeit low in yield (20%). Oxidations of thioether-S and NO2(-) were monitored by UV-Vis spectroscopy. Recovery of the sulfur oxidized ligands from their metal complexes allowed for their characterization by elemental analysis, (1)H NMR, FTIR and mass spectrometry. PMID:26390838

  20. Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis.

    PubMed

    Taylor, P; Hosea, N A; Tsigelny, I; Radić, Z; Berman, H A

    1997-01-01

    Acetylcholinesterase, an enzyme of the serine hydrolase family, catalyzes the rapid hydrolysis of certain carboxyl esters. Other acyl esters efficiently transacylate the enzyme with a subsequent, slow deacylation step. Of these, the phosphoryl and phosphonyl esters are perhaps of greatest mechanistic interest since individual enantiomers of known absolute stereochemistry can be isolated and their interactions with the dissymmetric enzyme active site examined. We describe here studies of a series of enantiomeric Rp- and Sp-alkylphosphonates interacting with mouse acetylcholinesterase. Since the acetylcholinesterase is generated by recombinant DNA methods, mutant enzymes can be made with specific replacements of individual amino acid side chains. Individual amino acid replacements in the acyl pocket, the choline subsite and at the active center gorge entry have been generated, and the reaction kinetics of the mutant enzymes analyzed. These studies have shown that substitution of aliphatic amino acids for phenylalanines 295 and 297 in the acyl pocket diminishes, and in some cases, actually inverts chiral preferences. The combined structure-activity approach, where both ligand and enzyme are modified systematically, has enabled us to show that the restricted dimensions of the acyl pocket in the active center dictate enantiomeric selectivity. Moreover, the reactions of compounds of known absolute stereochemistry show three requirements for efficient transphosphonylation: (a) apposition of the phosphate with the gamma-oxygen on Ser 203 to form a pentavalent, presumed trigonal bipyramidal intermediate, (b) polarization of the phosphonyl oxygen bond by its positioning in the oxyanion hole, and (c) positioning the leaving group towards the gorge exit. PMID:9676269

  1. Ligand-Enabled meta-C–H Activation Using a Transient Mediator

    PubMed Central

    Wang, Xiao-Chen; Gong, Wei; Fang, Li-Zhen; Zhu, Ru-Yi; Li, Suhua; Engle, Keary M.; Yu, Jin-Quan

    2015-01-01

    Achieving site selectivity in C–H functionalization reactions is a significant challenge, especially when the target C–H bond is distant from existing functional groups.1–5 Coordination of a functional group to a metal catalyst is often a key driving force and control element in many important reactions including asymmetric hydrogenation,6 epoxidation7, 8, and lithiation9. Exploitation of this effect has led to the development of a broad range of directed C–H activation reactions.10–14 However, such C–H activation methods are limited to proximal C–H bonds, which are spatially and geometrically accessible from the directing functional group. Development of meta-selective C–H functionalizations remains a significant challenge.1–5,15–17 We recently developed a U- shaped template that can be used to overcome this constraint and have shown that it can be used to selectively activate remote meta-C–H bonds.1, 2 While this approach has proven applicable for a diverse set of substrates and catalytic transformations,3–5 the need for a covalently attached complex template is a significant drawback for synthetic applications. In this manuscript, we report an alternative approach, one that employs norbornene as a transient mediator to achieve meta-selective C–H activation with a simple and common ortho-directing group. The use of a newly developed pyridine-based ligand is crucial for relaying the palladium catalyst to the meta position by norbornene following initial ortho- C–H activation. Thus, this catalytic reaction demonstrates the feasibility of switching ortho-selectivity to meta-selectivity in C–H activation of the same substrate by catalyst control. PMID:25754328

  2. Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity

    PubMed Central

    Adhireksan, Zenita; Davey, Gabriela E.; Campomanes, Pablo; Groessl, Michael; Clavel, Catherine M.; Yu, Haojie; Nazarov, Alexey A.; Yeo, Charmian Hui Fang; Ang, Wee Han; Dröge, Peter; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2014-01-01

    Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favourable toxicity and clearance properties. Nonetheless, their molecular targeting and mechanism of action are poorly understood. Here we study two prototypical ruthenium-arene agents—the cytotoxic antiprimary tumour compound [(η6-p-cymene)Ru(ethylene-diamine)Cl]PF6 and the relatively non-cytotoxic antimetastasis compound [(η6-p-cymene)Ru(1,3,5-triaza-7-phosphaadamantane)Cl2]—and discover that the former targets the DNA of chromatin, while the latter preferentially forms adducts on the histone proteins. Using a novel ‘atom-to-cell’ approach, we establish the basis for the surprisingly site-selective adduct formation behaviour and distinct cellular impact of these two chemically similar anticancer agents, which suggests that the cytotoxic effects arise largely from DNA lesions, whereas the protein adducts may be linked to the other therapeutic activities. Our study shows promise for developing new ruthenium drugs, via ligand-based modulation of DNA versus protein binding and thus cytotoxic potential, to target distinguishing epigenetic features of cancer cells. PMID:24637564

  3. Screening Outside the Catalytic Site: Inhibition of Macromolecular Inter-actions Through Structure-Based Virtual Ligand Screening Experiments

    PubMed Central

    Sperandio, Olivier; Miteva, Maria A; Segers, Kenneth; Nicolaes, Gerry A. F; Villoutreix, Bruno O

    2008-01-01

    During these last 15 years, drug discovery strategies have essentially focused on identifying small molecules able to inhibit catalytic sites. However, other mechanisms could be targeted. Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity. Along the same line, inhibition of protein-membrane could be of major importance in several disease indications. Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years. Importantly, through the existence of protein hot-spots and the presence of druggable pockets at the macromolecular interfaces or in their vicinities, it has been possible to find small molecule effectors using a variety of screening techniques, including combined virtual ligand-in vitro screening strategy. Indeed such in silico-in vitro protocols emerge as the method of choice to facilitate our quest of novel drug-like compounds or of mechanistic probes aiming at facilitating the understanding of molecular reactions involved in the Health and Disease process. In this review, we comment recent successes of combined in silico-in vitro screening methods applied to modulating macromolecular interactions with a special emphasis on protein-membrane interactions. PMID:18949072

  4. Structural characteristics of the recognition site for cholinergic ligands in the nicotinic acetylcholine receptor from squid optical ganglia

    SciTech Connect

    Plyashkevich, Yu.G.; Demushkin, V.P.

    1986-01-20

    The influence of chemical modification on the parameters of the binding of cholinergic ligands by the nicotinic acetylcholine receptor of squid optical ganglia was investigated. The presence of two subpopulations of recognition sites, differing in the composition of the groups contained in them, was detected. It was established with high probability that subpopulation I contains arginine and tyrosine residues and a carboxyl group while subpopulation II contains an amino group, a thyrosine residue, and a carboxyl group. Moreover, in both subpopulations there is an amino group important only for the binding of tubocurarin. On the basis of the results obtained, a model of the recognition sites for cholinergic ligands of the nicotinic acetylcholine receptor of squid optical ganglia is proposed.

  5. The utility of geometrical and chemical restraint information extracted from predicted ligand-binding sites in protein structure refinement.

    PubMed

    Brylinski, Michal; Lee, Seung Yup; Zhou, Hongyi; Skolnick, Jeffrey

    2011-03-01

    Exhaustive exploration of molecular interactions at the level of complete proteomes requires efficient and reliable computational approaches to protein function inference. Ligand docking and ranking techniques show considerable promise in their ability to quantify the interactions between proteins and small molecules. Despite the advances in the development of docking approaches and scoring functions, the genome-wide application of many ligand docking/screening algorithms is limited by the quality of the binding sites in theoretical receptor models constructed by protein structure prediction. In this study, we describe a new template-based method for the local refinement of ligand-binding regions in protein models using remotely related templates identified by threading. We designed a Support Vector Regression (SVR) model that selects correct binding site geometries in a large ensemble of multiple receptor conformations. The SVR model employs several scoring functions that impose geometrical restraints on the Cα positions, account for the specific chemical environment within a binding site and optimize the interactions with putative ligands. The SVR score is well correlated with the RMSD from the native structure; in 47% (70%) of the cases, the Pearson's correlation coefficient is >0.5 (>0.3). When applied to weakly homologous models, the average heavy atom, local RMSD from the native structure of the top-ranked (best of top five) binding site geometries is 3.1Å (2.9Å) for roughly half of the targets; this represents a 0.1 (0.3)Å average improvement over the original predicted structure. Focusing on the subset of strongly conserved residues, the average heavy atom RMSD is 2.6Å (2.3Å). Furthermore, we estimate the upper bound of template-based binding site refinement using only weakly related proteins to be ∼2.6Å RMSD. This value also corresponds to the plasticity of the ligand-binding regions in distant homologues. The Binding Site Refinement (BSR

  6. Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination

    NASA Astrophysics Data System (ADS)

    Heath, Brittany L.; Jockusch, Rebecca A.

    2012-11-01

    Knowledge of the structure of protein-ligand complexes can aid in understanding their roles within complex biological processes. Here we use electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer to investigate the noncovalent binding of the macrocycle cucurbit[7]uril (CB7) to bovine insulin. Recent condensed-phase experiments (Chinai et al., J. Am. Chem. Soc. 133:8810-8813, 2011) indicate that CB7 binds selectively to the N-terminal phenylalanine of the insulin B-chain. Competition experiments employing ESI mass spectrometry to assess complex formation between CB7 and wild type insulin B-chain vs. a mutant B-chain, confirm that the N-terminal phenylalanine plays in important role in solution-phase binding. However, analysis of fragment ions produced by electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the insulin B-chain suggests a different picture. The apparent gas-phase binding site, as identified by the ECD, lies further along the insulin B-chain. Together, these studies thus indicate that the CB7 ligand migrates in the ESI mass spectrometry analysis. Migration is likely aided by the presence of additional interactions between CB7 and the insulin B-chain, which are not observed in the crystal structure. While this conformational difference may result simply from the removal of solvent and addition of excess protons by the ESI, we propose that the migration may be enhanced by charge reduction during the ECD process itself because ion-dipole interactions are key to CB7 binding. The results of this study caution against using ECD-MS as a stand-alone structural probe for the determination of solution-phase binding sites.

  7. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  8. Synthesis, Characterization, and Reactivity Studies of Iron Complexes Supported by the Redox-Active [ONO] Ligand

    NASA Astrophysics Data System (ADS)

    Wong, Janice Lin

    The work reported herein primarily focuses on the development of new platforms for multi-electron reactivity using iron complexes supported by a redox-active pincer-type ligand. This dissertation details the synthesis, characterization, and reactivity of iron complexes coordinated to the redox-active [ONO] ([ONO]H3 = bis(3,5-di-tert-butyl-2-phenol)amine) ligand. Chapter 1 provides a general background on ligand-centered and metal-centered redox reactivity. Specifically, the characteristics of redox-active ligands and their ability to promote multi-electron reactivity at redox-inert metal centers is presented. In addition, iron-catalyzed organic transformations in which the metal center undergoes redox changes is also discussed. Finally, ligand-enabled redox reactions mediated by iron complexes containing redox-active ligands is described. Chapter 2 reports on the complexation of bis(3,5-di-tert-butyl-2-phenoxy)amine, [ONHO], and the redox-active [ONO] ligands by iron centers to afford a new family of iron complexes. Characterizations of each compound through a battery of analytical techniques reveal the oxidation states of the metal center and ligand. Furthermore, the electronic properties of each complex were investigated in order to evaluate their potential to facilitate multi-electron reactivity. Chapter 3 details the reactivity of the [ONO]Fe platform. Metathesis reactions are conducted with [ONOq]FeIIIX 2 (X = Cl, N[SiMe3]2) complexes, demonstrating the capability of the fully-oxidized [ONOq]1-- to act as a two-electron acceptor to generate the fully reduced [ONO cat]3-- that is coordinated to an iron(III) center. Similarly, oxidation of [ONOcat]FeIII(py) 3 (py = pyridine) using dihalogens result in two-electron oxidations of the tridentate ligand while the metal oxidation state remains the same. These redox reactions showcase the ability of the [ONO] ligand platform to undergo reversible two-electron oxidation state changes, allowing multi-electron reactivity

  9. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  10. Peroxisome Proliferator-Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies.

    PubMed

    Yousefi, Bahman; Samadi, Nasser; Baradaran, Behzad; Shafiei-Irannejad, Vahid; Zarghami, Nosratollah

    2016-07-01

    Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail. PMID:26841308

  11. Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

    PubMed Central

    Watanabe, H; Tanase, S; Nakajou, K; Maruyama, T; Kragh-Hansen, U; Otagiri, M

    2000-01-01

    Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411. PMID:10903143

  12. Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

    PubMed

    Watanabe, H; Tanase, S; Nakajou, K; Maruyama, T; Kragh-Hansen, U; Otagiri, M

    2000-08-01

    Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411. PMID:10903143

  13. Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration.

    PubMed

    Schulte, Carsten; Ferraris, Gian Maria Sarra; Oldani, Amanda; Galluzzi, Massimiliano; Podestà, Alessandro; Puricelli, Luca; de Lorenzi, Valentina; Lenardi, Cristina; Milani, Paolo; Sidenius, Nicolai

    2016-01-01

    The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required. PMID:26616200

  14. Ligand determinants of nisin for its induction activity.

    PubMed

    Ge, Xiaoxuan; Teng, Kunling; Wang, Jian; Zhao, Fangyuan; Wang, Fangfang; Zhang, Jie; Zhong, Jin

    2016-07-01

    Nisin has been widely used in the food industry as a safe and natural preservative and has the potential to be used as a biomedicine. Improving nisin production is important for its enormous applications. Nisin A is produced in Lactococcus lactis and its biosynthesis is induced through the regulation of the 2-component system NisKR. In this study, alanine-scanning mutagenesis was applied to study the key structure or AA in nisin for inducing the 2-component system NisKR to regulate downstream gene expression. Assay of β-galactosidase activity revealed that either ring A or ring B was necessary for nisin to induce lacZ reporter gene expression. A substituted first ring formed by Thr2 and Cys7 in S3A instead of ring A (formed by Ser3 and Cys7) fully retained nisin induction activity. Mutation of cationic AA and addition of cationic ions hardly affected nisin induction activity. These results demonstrated that the N-terminal ring structures in nisin were involved in activating NisKR to act as an inducing molecule, whereas the electrostatic force might not contribute to this process. In addition, 2 specific residues were revealed to have potential for improving both nisin induction and antimicrobial activity, which might be used for increasing nisin production. PMID:27132090

  15. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  16. Synthesis and anti-fungicidal activity of some transition metal complexes with benzimidazole dithiocarbamate ligand

    NASA Astrophysics Data System (ADS)

    Mohamed, Gehad G.; Ibrahim, Nasser A.; Attia, Hanaa A. E.

    2009-04-01

    Seven transition metal complexes of benzimidazole ligand (HL) are reported and characterized based on elemental analyses, IR, solid reflectance, magnetic moment, molar conductance and thermal analyses (TGA and DTA). From the obtained data, the complexes were proposed to have the general formulae [MX 2(HL)(H 2O)]· yH 2O, where M = Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cr(III); X = Cl -, SO 42- and y = 0-4. The molar conductance data revealed that all the metal chelates were non-electrolytes. From the magnetic and solid reflectance spectra, it was found that the geometrical structure of these complexes is octahedral. The thermal behaviour of these chelates showed that the hydrated complexes loss water molecules of hydration in the first step followed immediately by decomposition of the anions and ligand molecules in the subsequent steps. Fungicidal activity of the prepared complexes and free ligand was evaluated against three soil borne fungi. Data obtained showed the higher biological activity of the prepared complexes than the parent Schiff base ligand. Formulation of the most potent complex was carried out in the form of 25% WP. Fungicidal activity of the new formulation was evaluated and compared with the standard fungicide Pencycuron (Monceren 25% WP). In most cases, the new formulation possessed higher fungicidal activity than the standard fungicide under the laboratory conditions.

  17. Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors.

    PubMed

    Selvaraj, Chandrabose; Omer, Ankur; Singh, Poonam; Singh, Sanjeev Kumar

    2015-01-01

    Retroviruses HIV-1 and HTLV-1 are chiefly considered to be the most dangerous pathogens in Homo sapiens. These two viruses have structurally unique protease (PR) enzymes, which are having common function of its replication mechanism. Though HIV PR drugs failed to inhibit HTLV-1 infections, they emphatically emphasise the need for designing new lead compounds against HTLV-1 PR. Therefore, we tried to understand the binding level interactions through the charge environment present in both ligand and protein active sites. The domino effect illustrates that libraries of purvalanol-A are attuned to fill allosteric binding site of HTLV-1 PR through molecular recognition and shows proper binding of ligand pharmacophoric features in receptor contours. Our screening evaluates seven compounds from purvalanol-A libraries, and these compounds' pharmacophore searches for an appropriate place in the binding site and it places well according to respective receptor contour surfaces. Thus our result provides a platform for the progress of more effective compounds, which are better in free energy calculation, molecular docking, ADME and molecular dynamics studies. Finally, this research provided novel chemical scaffolds for HTLV-1 drug discovery. PMID:25335799

  18. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion.

    PubMed

    Zarkesh, Ryan A; Ichimura, Andrew S; Monson, Todd C; Tomson, Neil C; Anstey, Mitchell R

    2016-06-14

    The redox-active bis(imino)acenapthene (BIAN) ligand was used to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events. PMID:26998892

  19. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion

    DOE PAGESBeta

    Zarkesh, Ryan A.; Ichimura, Andrew S.; Monson, Todd C.; Tomson, Neil C.; Anstey, Mitchell R.

    2016-02-01

    We used the redox-active bis(imino)acenapthene (BIAN) ligand to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Ultimately, complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events.

  20. Homotropic cooperativity from the activation pathway of the allosteric ligand-responsive regulatory trp RNA-binding attenuation protein.

    PubMed

    Kleckner, Ian R; McElroy, Craig A; Kuzmic, Petr; Gollnick, Paul; Foster, Mark P

    2013-12-10

    The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two-binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that dissociation of Trp from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature-dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. PMID:24224873

  1. The Novel Dipeptide Translocator Protein Ligand, Referred to As GD-23, Exerts Anxiolytic and Nootropic Activities

    PubMed Central

    Povarnina, P. Yu.; Yarkov, S. A.; Gudasheva, T. A.; Yarkova, M. A.; Seredenin, S. B.

    2015-01-01

    The translocator protein (TSPO) promotes the translocation of cholesterol to the inner mitochondrial membrane and mediates steroid formation. In this study, we first report on a biological evaluation of the dipeptide GD-23 (N-carbobenzoxy-L tryptophanyl-L isoleucine amide), a structural analogue of Alpidem, the principal TSPO ligand. We show that GD-23 in a dose range of 0.05 to 0.5 mg/kg (i.p.) exhibits anxiolytic activity in the elevated plus maze test and nootropic activity in the object recognition test in scopolamine-induced amnesia in rodents. It was shown that GD-23 did not affect spontaneous locomotor activity, holding promise as a nonsedative anxiolytic agent. The anxiolytic and nootropic activities of GD-23 were abrogated by the TSPO specific ligand PK11195, which thus suggests a role for TSPO in mediating the pharmacological activity of GD-23. PMID:26483966

  2. The Novel Dipeptide Translocator Protein Ligand, Referred to As GD-23, Exerts Anxiolytic and Nootropic Activities.

    PubMed

    Povarnina, P Yu; Yarkov, S A; Gudasheva, T A; Yarkova, M A; Seredenin, S B

    2015-01-01

    The translocator protein (TSPO) promotes the translocation of cholesterol to the inner mitochondrial membrane and mediates steroid formation. In this study, we first report on a biological evaluation of the dipeptide GD-23 (N-carbobenzoxy-L tryptophanyl-L isoleucine amide), a structural analogue of Alpidem, the principal TSPO ligand. We show that GD-23 in a dose range of 0.05 to 0.5 mg/kg (i.p.) exhibits anxiolytic activity in the elevated plus maze test and nootropic activity in the object recognition test in scopolamine-induced amnesia in rodents. It was shown that GD-23 did not affect spontaneous locomotor activity, holding promise as a nonsedative anxiolytic agent. The anxiolytic and nootropic activities of GD-23 were abrogated by the TSPO specific ligand PK11195, which thus suggests a role for TSPO in mediating the pharmacological activity of GD-23. PMID:26483966

  3. The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

    PubMed

    Gudasheva, T A; Deeva, O A; Mokrov, G V; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-01-01

    On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action. PMID:26518550

  4. Design, Synthesis, and Structure–Activity Relationships of Highly Potent 5-HT3 Receptor Ligands

    PubMed Central

    2012-01-01

    The 5-HT3 receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT3R hit fragment. Here we describe the synthesis and structure–activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT3R affinity using a [3H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pKi > 10) for the 5-HT3 receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT3 ligands and is used for ligand–receptor binding mode prediction using homology modeling and in silico docking approaches. PMID:23006041

  5. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway.

    PubMed

    Okahashi, Nobuo; Inaba, Hiroaki; Nakagawa, Ichiro; Yamamura, Taihei; Kuboniwa, Masae; Nakayama, Koji; Hamada, Shigeyuki; Amano, Atsuo

    2004-03-01

    Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production. PMID:14977979

  6. Metal Complexes of Macrocyclic Schiff-Base Ligand: Preparation, Characterisation, and Biological Activity

    PubMed Central

    Ahmed, Riyadh M.; Yousif, Enaam I.; Hasan, Hasan A.; Al-Jeboori, Mohamad J.

    2013-01-01

    A new macrocyclic multidentate Schiff-base ligand Na4L consisting of two submacrocyclic units (10,21-bis-iminomethyl-3,6,14,17-tricyclo[17.3.1.18,12]tetracosa-1(23),2,6,8,10,12(24),13,17,19,21,-decaene-23,24-disodium) and its tetranuclear metal complexes with Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) are reported. Na4L was prepared via a template approach, which is based on the condensation reaction of sodium 2,4,6-triformyl phenolate with ethylenediamine in mole ratios of 2 : 3. The tetranuclear macrocyclic-based complexes were prepared from the reaction of the corresponding metal chloride with the ligand. The mode of bonding and overall geometry of the compounds were determined through physicochemical and spectroscopic methods. These studies revealed tetrahedral geometries about Mn, Co, and Zn atoms. However, square planar geometries have been suggested for NiII and CuII complexes. Biological activity of the ligand and its metal complexes against Gram positive bacterial strain Staphylococcus aureus and Gram negative bacteria Escherichia coli revealed that the metal complexes become more potentially resistive to the microbial activities as compared to the free ligand. However, these metal complexes do not exhibit any effects on the activity of Pseudomonas aeruginosa bacteria. There is therefore no inhibition zone. PMID:23935414

  7. Peroxisome proliferator-activated receptor ligands as antiatherogenic agents: panacea or another Pandora's box?

    PubMed

    Molavi, Behzad; Rasouli, Neda; Mehta, Jawahar L

    2002-01-01

    Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor super family that modulate gene expression upon ligand activation. They are 3 major subtypes of PPARs: alpha, delta (also called beta), and gamma. PPAR-gamma is widely expressed in the cardiovascular system and is involved in the regulation of tissue inflammation and smooth muscle cell growth pathways as well as in lipoprotein metabolism and coagulation cascades. PPAR-gamma ligands of (e.g., rosigitazone and pioglitazone) have been shown to exert antiatherogenic effects both in vitro and in vivo. PPAR-alpha ligands (e.g., clofibrate and benzofibrate) modulate lipoprotein metabolism, and affect inflammation and coagulation cascade. These effects may be helpful in resolving the dilemma arising from studies that showed significant mortality and morbidity benefits of fibrates in the face of minimal changes in HDL-cholesterol levels. The role of PPAR-delta in atherogenesis remains largely unknown, although it appears that PPAR-delta activation affects lipoprotein metabolism. PPAR ligands appear to be promising agents in limiting atherosclerosis; however, large-scale clinical trials are required to assess their safety and efficacy before they can be added to the clinicians' arsenal of antiatherosclerotic agents. PMID:12000972

  8. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  9. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  10. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    SciTech Connect

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  11. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

    PubMed Central

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

    2015-01-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that

  12. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility.

    PubMed

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S; Olson, Arthur J; Sanner, Michel F

    2015-12-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR-AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 -a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 -a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down

  13. A robust and efficient algorithm for the shape description of protein structures and its application in predicting ligand binding sites

    PubMed Central

    Xie, Lei; Bourne, Philip E

    2007-01-01

    Background An accurate description of protein shape derived from protein structure is necessary to establish an understanding of protein-ligand interactions, which in turn will lead to improved methods for protein-ligand docking and binding site analysis. Most current shape descriptors characterize only the local properties of protein structure using an all-atom representation and are slow to compute. We need new shape descriptors that have the ability to capture both local and global structural information, are robust for application to models and low quality structures and are computationally efficient to permit high throughput analysis of protein structures. Results We introduce a new shape description that requires only the Cα atoms to represent the protein structure, thus making it both fast and suitable for use on models and low quality structures. The notion of a geometric potential is introduced to quantitatively describe the shape of the structure. This geometric potential is dependent on both the global shape of the protein structure as well as the surrounding environment of each residue. When applying the geometric potential for binding site prediction, approximately 85% of known binding sites can be accurately identified with above 50% residue coverage and 80% specificity. Moreover, the algorithm is fast enough for proteome-scale applications. Proteins with fewer than 500 amino acids can be scanned in less than two seconds. Conclusion The reduced representation of the protein structure combined with the geometric potential provides a fast, quantitative description of protein-ligand binding sites with potential for use in large-scale predictions, comparisons and analysis. PMID:17570152

  14. Evidence for Ligand-Independent Activation of Hippocampal Estrogen Receptor-α by IGF-1 in Hippocampus of Ovariectomized Rats.

    PubMed

    Grissom, Elin M; Daniel, Jill M

    2016-08-01

    In the absence of ovarian estrogens, increased levels of estrogen receptor (ER)α in the hippocampus are associated with improvements in cognition. In vitro evidence indicates that under conditions of low estrogen, growth factors, including Insulin-Like Growth Factor 1 (IGF-1), can activate ERα and regulate ERα-mediated transcription through mechanisms that likely involve modification of phosphorylation sites on the receptor. The goal of the current work was to investigate a role for IGF-1 in ligand-independent activation of ERα in the hippocampus of female rats. Ovariectomized rats received a single intracerebroventricular infusion of IGF-1 and hippocampi were collected 1 or 24 hours later. After 1 h, IGF-1 increased hippocampal levels of phosphorylated ERα at serine 118 (S118) as revealed by Western blotting. Coimmunoprecipitation revealed that at 1 hour after infusion, IGF-1 increased association between ERα and steroid receptor coactivator 1, a histone acetyltransferase that increases transcriptional activity of phosphorylated ERα. IGF-1 infusion increased levels of the ERα-regulated proteins ERα, choline acetyltransferase, and brain-derived neurotrophic factor in the hippocampus 24 hours after infusion. Results indicate that IGF-1 activates ERα in ligand-independent manner in the hippocampus via phosphorylation at S118 resulting in increased association of ERα with steroid receptor coactivator 1 and elevation of ER-regulated proteins. To our knowledge, these data are the first in vivo evidence of ligand-independent actions of ERα and provide a mechanism by which ERα can impact memory in the absence of ovarian estrogens. PMID:27254005

  15. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  16. Improving the performance of the PLB index for ligand-binding site prediction using dihedral angles and the solvent-accessible surface area.

    PubMed

    Cao, Chen; Xu, Shutan

    2016-01-01

    Protein ligand-binding site prediction is highly important for protein function determination and structure-based drug design. Over the past twenty years, dozens of computational methods have been developed to address this problem. Soga et al. identified ligand cavities based on the preferences of amino acids for the ligand-binding site (RA) and proposed the propensity for ligand binding (PLB) index to rank the cavities on the protein surface. However, we found that residues exhibit different RAs in response to changes in solvent exposure. Furthermore, previous studies have suggested that some dihedral angles of amino acids in specific regions of the Ramachandran plot are preferred at the functional sites of proteins. Based on these discoveries, the amino acid solvent-accessible surface area and dihedral angles were combined with the RA and PLB to obtain two new indexes, multi-factor RA (MF-RA) and multi-factor PLB (MF-PLB). MF-PLB, PLB and other methods were tested using two benchmark databases and two particular ligand-binding sites. The results show that MF-PLB can improve the success rate of PLB for both ligand-bound and ligand-unbound structures, particularly for top choice prediction. PMID:27619067

  17. Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    PubMed Central

    Valley, Christopher C.; Arndt-Jovin, Donna J.; Karedla, Narain; Steinkamp, Mara P.; Chizhik, Alexey I.; Hlavacek, William S.; Wilson, Bridget S.; Lidke, Keith A.; Lidke, Diane S.

    2015-01-01

    Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization. PMID:26337388

  18. Syntheses, characterization, biological activities and photophysical properties of lanthanides complexes with a tetradentate Schiff base ligand.

    PubMed

    Taha, Ziyad A; Ajlouni, Abdulaziz M; Al Momani, Waleed; Al-Ghzawi, Abeer A

    2011-10-15

    A tetradentate Schiff base ligand L (N,N'-bis(1-naphthaldimine)-o-phenylenediamine) was prepared from the condensation of 2-hydroxy-1-naphthaldehyde with o-phenylenediamine in a molar ratio of 2:1. New eight lanthanide metal complexes [LnL(NO(3))(2)(H(2)O)(x)](NO(3)) {Ln(III) = Nd, Dy, Sm, Pr, Gd, Tb, La and Er, x = 0 for Nd, Sm, 1 for La, Gd, Pr, Nd, Dy, and 2 for Tb} were prepared. The characterization and nature of bonding of these complexes were elucidated by elemental analysis, spectral analysis ((1)H NMR, FT-IR, UV-vis), molar conductivity measurements, luminescence spectra and thermogravimetric studies. Analytical and spectral data revealed that the ligand L coordinates to the central Ln(III) ions by its two imine nitrogen atoms and two phenolic oxygen atoms with 1:1 stoichiometry. Under the excitation with 329 nm at room temperature, Tb and Dy complexes exhibited characteristic luminescence of the central metal ions attributed to efficient energy transfer from the ligand to the metal center. Most of Ln(III) complexes found to exhibit antibacterial activities against a number of pathogenic bacteria. We found that the antioxident activity of Ln(III) complexes on DPPH(•) is concentration dependent and higher than that of the free ligand L. PMID:21764359

  19. Molecular Fingerprint-based Artificial Neural Networks QSAR for Ligand Biological Activity Predictions

    PubMed Central

    Myint, Kyaw-Zeyar; Wang, Lirong; Tong, Qin; Xie, Xiang-Qun

    2012-01-01

    In this manuscript, we have reported a novel 2D fingerprint-based artificial neural network QSAR (FANN-QSAR) method in order to effectively predict biological activities of structurally diverse chemical ligands. Three different types of fingerprints, namely ECFP6, FP2 and MACCS, were used in FANN-QSAR algorithm development, and FANN-QSAR models were compared to known 3D and 2D QSAR methods using five data sets previously reported. In addition, the derived models were used to predict GPCR cannabinoid ligand binding affinities using our manually curated cannabinoid ligand database containing 1699 structurally diverse compounds with reported cannabinoid receptor subtype CB2 activities. To demonstrate its useful applications, the established FANN-QSAR algorithm was used as a virtual screening tool to search a large NCI compound database for lead cannabinoid compounds and we have discovered several compounds with good CB2 binding affinities ranging from 6.70 nM to 3.75 μM. To the best of our knowledge, this is the first report for a fingerprint-based neural network approach validated with a successful virtual screening application in identifying lead compounds. The studies proved that the FANN-QSAR method is a useful approach to predict bioactivities or properties of ligands and to find novel lead compounds for drug discovery research. PMID:22937990

  20. Bivalent transition metal complexes of ONO donor hydrazone ligand: Synthesis, structural characterization and antimicrobial activity.

    PubMed

    Bhaskar, Ravindra; Salunkhe, Nilesh; Yaul, Amit; Aswar, Anand

    2015-12-01

    Mononuclear transition metal complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with a new hydrazone ligand derived from pyrazine-2-carbohydrazide and 2-hydroxyacetophenone have been synthesized. The isolated complexes were characterized by elemental analysis, spectral and analytical methods including elemental analyses, IR, diffuse reflectance, (1)H-NMR, mass spectra, molar conductance, magnetic moment, ESR, XRD, TG and SEM analysis. From the elemental analyses data, the stoichiometry of the complexes was found to be 1:1 (metal:ligand) having the general formulae [M(HL)(Cl)(H2O)2], [M=Mn(II), Co(II), Ni(II) and Cu(II)] and [M(L)(H2O)], [M=Zn(II) and Cd(II)]. The molar conductance values indicate the nonelectrolytic nature of metal complexes. The IR spectral data suggest that the ligand behaves as tridentate moiety with ONO donor atoms sequence towards central metal ion. The Mn(II), Co(II), Ni(II) and Cu(II) complexes have been assigned a monomeric octahedral geometry whereas tetrahedral to Zn(II) and Cd(II) complexes. The antibacterial and antifungal activities of the ligand and its metal complexes were studied against bacterial species Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and Streptococcus pyogenes and fungi Candida albicans, Aspergillus niger and Aspergillus clavatus. The activity data show that the metal complexes have a promising biological activity comparable with the parent ligand against all bacterial and fungal species. PMID:26163785

  1. Ligand Binding and Activation of PPARγ by Firemaster® 550: Effects on Adipogenesis and Osteogenesis in Vitro

    PubMed Central

    Pillai, Hari K.; Fang, Mingliang; Beglov, Dmitri; Kozakov, Dima; Vajda, Sandor; Stapleton, Heather M.; Webster, Thomas F.

    2014-01-01

    Background: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents. Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP. Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously. Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP. Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550. Citation: Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ. 2014

  2. Rational Quantitative Structure-Activity Relationship (RQSAR) Screen for PXR and CAR Isoform-Specific Nuclear Receptor Ligands

    PubMed Central

    Dring, Ann M.; Anderson, Linnea E.; Qamar, Saima; Stoner, Matthew A.

    2010-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets. PMID:20869355

  3. Highly potent, non-basic 5-HT6 ligands. Site mutagenesis evidence for a second binding mode at 5-HT6 for antagonism.

    PubMed

    Harris, Ralph N; Stabler, Russel S; Repke, David B; Kress, James M; Walker, Keith A; Martin, Renee S; Brothers, Julie M; Ilnicka, Mariola; Lee, Simon W; Mirzadegan, Tara

    2010-06-01

    A series of 5-HT(6) ligands derived from (R)-1-(amino)methyl-6-(phenyl)sulfonyltetralin was prepared that yielded several non-basic analogs having sub-nanomolar affinity. Ligand structure-activity relationships, receptor point mutation studies, and molecular modeling of these novel ligands all combined to reveal a new alternative binding mode to 5-HT(6) for antagonism. PMID:20434910

  4. Constructing query-driven dynamic machine learning model with application to protein-ligand binding sites prediction.

    PubMed

    Yu, Dong-Jun; Hu, Jun; Li, Qian-Mu; Tang, Zhen-Min; Yang, Jing-Yu; Shen, Hong-Bin

    2015-01-01

    We are facing an era with annotated biological data rapidly and continuously generated. How to effectively incorporate new annotated data into the learning step is crucial for enhancing the performance of a bioinformatics prediction model. Although machine-learning-based methods have been extensively used for dealing with various biological problems, existing approaches usually train static prediction models based on fixed training datasets. The static approaches are found having several disadvantages such as low scalability and impractical when training dataset is huge. In view of this, we propose a dynamic learning framework for constructing query-driven prediction models. The key difference between the proposed framework and the existing approaches is that the training set for the machine learning algorithm of the proposed framework is dynamically generated according to the query input, as opposed to training a general model regardless of queries in traditional static methods. Accordingly, a query-driven predictor based on the smaller set of data specifically selected from the entire annotated base dataset will be applied on the query. The new way for constructing the dynamic model enables us capable of updating the annotated base dataset flexibly and using the most relevant core subset as the training set makes the constructed model having better generalization ability on the query, showing "part could be better than all" phenomenon. According to the new framework, we have implemented a dynamic protein-ligand binding sites predictor called OSML (On-site model for ligand binding sites prediction). Computer experiments on 10 different ligand types of three hierarchically organized levels show that OSML outperforms most existing predictors. The results indicate that the current dynamic framework is a promising future direction for bridging the gap between the rapidly accumulated annotated biological data and the effective machine-learning-based predictors. OSML

  5. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.

    PubMed

    Nguyen, Duy P; Miyaoka, Yuichiro; Gilbert, Luke A; Mayerl, Steven J; Lee, Brian H; Weissman, Jonathan S; Conklin, Bruce R; Wells, James A

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  6. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

    PubMed Central

    Nguyen, Duy P.; Miyaoka, Yuichiro; Gilbert, Luke A.; Mayerl, Steven J.; Lee, Brian H.; Weissman, Jonathan S.; Conklin, Bruce R.; Wells, James A.

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  7. Structure-Based Identification of Novel Ligands Targeting Multiple Sites within a Chemokine-G-Protein-Coupled-Receptor Interface.

    PubMed

    Smith, Emmanuel W; Nevins, Amanda M; Qiao, Zhen; Liu, Yan; Getschman, Anthony E; Vankayala, Sai L; Kemp, M Trent; Peterson, Francis C; Li, Rongshi; Volkman, Brian F; Chen, Yu

    2016-05-12

    CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches. PMID:27058821

  8. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  9. Computational design of an endo-1,4-β-xylanase ligand binding site

    PubMed Central

    Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie; Harp, Joel M.; Mizoue, Laura S.; Meiler, Jens

    2011-01-01

    The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein–ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-β-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the ‘thumb’ region of our design scaffold intrinsic to the family 11 β-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the β-xylanase proteins. PMID:21349882

  10. Computational design of an endo-1,4-[beta]-xylanase ligand binding site

    SciTech Connect

    Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie; Harp, Joel M.; Mizoue, Laura S.; Meiler, Jens

    2012-09-05

    The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.

  11. Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors.

    PubMed Central

    DiRenzo, J; Söderstrom, M; Kurokawa, R; Ogliastro, M H; Ricote, M; Ingrey, S; Hörlein, A; Rosenfeld, M G; Glass, C K

    1997-01-01

    As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation. PMID:9121466

  12. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  13. Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O2 Activation

    PubMed Central

    2015-01-01

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes. PMID

  14. Toxic and chemopreventive ligands preferentially activate distinct aryl hydrocarbon receptor pathways: implications for cancer prevention.

    PubMed

    Okino, Steven T; Pookot, Deepa; Basak, Shashwati; Dahiya, Rajvir

    2009-03-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that controls estrogen action through two distinct pathways. In one pathway, AhR acts as a transcription factor that induces the expression of the CYP1 family of estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation of the estrogen receptor and suppresses estrogen signaling. The AhR ligand 3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents breast tumors in rodents and is associated with decreased breast cancer risk in humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to gain insight into how two AhR ligands can exert such fundamentally different health effects. We find that DIM and TCDD have differing abilities to activate the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1 gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD has a notably weaker effect on these processes. Small interfering RNA knockdown of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our findings reveal that DIM and TCDD each elicit a unique pattern of change in pathways that control estrogen action; such patterns may determine if an AhR ligand has beneficial or adverse health effects. PMID:19223575

  15. Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization

    SciTech Connect

    Lai, W.H.; Cameron, P.H.; Doherty, J.J. II; Posner, B.I.; Bergeron, J.J. )

    1989-12-01

    The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.

  16. Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

    PubMed Central

    Fondell, J D; Ge, H; Roeder, R G

    1996-01-01

    Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8710870

  17. Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

    PubMed Central

    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

    2014-01-01

    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 ([125I]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 [125I]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. PMID:25179220

  18. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    PubMed

    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. PMID:25179220

  19. Ligand Biological Activity Predictions Using Fingerprint-Based Artificial Neural Networks (FANN-QSAR)

    PubMed Central

    Myint, Kyaw Z.; Xie, Xiang-Qun

    2015-01-01

    This chapter focuses on the fingerprint-based artificial neural networks QSAR (FANN-QSAR) approach to predict biological activities of structurally diverse compounds. Three types of fingerprints, namely ECFP6, FP2, and MACCS, were used as inputs to train the FANN-QSAR models. The results were benchmarked against known 2D and 3D QSAR methods, and the derived models were used to predict cannabinoid (CB) ligand binding activities as a case study. In addition, the FANN-QSAR model was used as a virtual screening tool to search a large NCI compound database for lead cannabinoid compounds. We discovered several compounds with good CB2 binding affinities ranging from 6.70 nM to 3.75 μM. The studies proved that the FANN-QSAR method is a useful approach to predict bioactivities or properties of ligands and to find novel lead compounds for drug discovery research. PMID:25502380

  20. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  1. A theoretical investigation of the functional role of the axial methionine ligand of the Cu(A) site in cytochrome c oxidase.

    PubMed

    Kang, Jiyoung; Kino, Hiori; Tateno, Masaru

    2011-10-01

    The functional roles of the amino acid residues of the Cu(A) site in bovine cytochrome c oxidase (CcO) were investigated by utilizing hybrid quantum mechanics (QM)/molecular mechanics (MM) calculations. The energy levels of the molecular orbitals (MOs) involving Cu d(zx) orbitals unexpectedly increased, as compared with those found previously with a simplified model system lacking the axial Met residue (i.e., Cu(2)S(2)N(2)). This elevation of MO energies stemmed from the formation of the anti-bonding orbitals, which are generated by hybridization between the d(zx) orbitals of Cu ions and the p-orbitals of the S and O atoms of the axial ligands. To clarify the roles of the axial Met ligand, the inner-sphere reorganization energies of the Cu(A) site were computed, with the Met residue assigned to either the QM or MM region. The reorganization energy slightly increased when the Met residue was excluded from the QM region. The existing experimental data and the present structural modeling study also suggested that the axial Met residue moderately increased the redox potential of the Cu(A) site. Thus, the role of the Met may be to regulate the electron transfer rate through the fine modulation of the electronic structure of the Cu(A) "platform", created by two Cys/His residues coordinated to the Cu ions. This regulation would provide the optimum redox potential/reorganization energy of the Cu(A) site, and thereby facilitate the subsequent cooperative reactions, such as the proton pump and the enzymatic activity, of CcO. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins. PMID:21745457

  2. Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase.

    PubMed

    Raasakka, Arne; Myllykoski, Matti; Laulumaa, Saara; Lehtimäki, Mari; Härtlein, Michael; Moulin, Martine; Kursula, Inari; Kursula, Petri

    2015-01-01

    2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release. PMID:26563764

  3. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    SciTech Connect

    Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

    2009-05-26

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  4. Investigation of the Copper Binding Site and the Role of Histidine as a Ligand in Riboflavin Binding Protein

    PubMed Central

    Smith, Sheila R.; Bencze, Krisztina Z.; Russ, Kristen A.; Wasiukanis, Kristen; Benore-Parsons, Marilee; Stemmler, Timothy L.

    2008-01-01

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 Å, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu–O3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit. PMID:18593109

  5. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

  6. In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.

    PubMed Central

    Iwama, T; Kawagishi, I; Gomi, S; Homma, M; Imae, Y

    1995-01-01

    The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein. PMID:7721714

  7. Molecular and Cellular Regulation of Toll-Like Receptor-4 Activity Induced by Lipopolysaccharide Ligands

    PubMed Central

    Liaunardy-Jopeace, Ardiyanto; Gay, Nicholas J.

    2014-01-01

    As well as being the primary signaling receptor for bacterial endotoxin or lipopolysaccharide Toll-like receptor-4 function is modulated by numerous factors not only in the context of microbial pathogenesis but also autoimmune and allergic diseases. TLR4 is subject to multiple levels of endogenous control and regulation from biosynthesis and trafficking to signal transduction and degradation. On the other hand regulation of TLR4 activity breaks down during Gram −ve sepsis leading to systemic damage, multi organ failure, and death. In this article, we review how TLR4 traffics from the early secretory pathway, the cis/trans Golgi to the cell surface and endolysosomal compartments. We will present evidence about how these processes influence signaling and can potentially lead to increased sensitivity to ligand-dependent activation as well as ligand-independent constitutive activation that may contribute to pathogenesis in sepsis. We will also discuss how sustained signaling may be coupled to endocytosis and consider the potential molecular mechanisms of immuno-modulators that modify TLR4 signaling function including the cat allergen FelD1 and endogenous protein ligands such as the extracellular matrix protein tenascin C and calprotectin (MRP8/14). PMID:25339952

  8. A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours.

    PubMed

    Li, Juan; Wang, Fengshan; Sun, Deqing; Wang, Rongmei

    2016-08-01

    It has been 30 years since the discovery of the anti-tumour property of paclitaxel (PTX), which has been successfully applied in clinic for the treatment of carcinomas of the lungs, breast and ovarian. However, PTX is poorly soluble in water and has no targeting and selectivity to tumour tissue. Recent advances in active tumour targeting of PTX delivery vehicles have addressed some of the issues related to lack of solubility in water and non-specific toxicities associated with PTX. These PTX delivery vehicles are designed for active targeting to specific cancer cells by the addition of ligands for recognition by specific receptors/antigens on cancer cells. This article will focus on various ligands and related targeting strategies serving as potential tools for active targeting of PTX to tumour tissues, illustrating their use in different tumour models. This review also highlights the need of further studies on the discovery of receptors in different cells of specific organ and ligands with binding efficiency to these specific receptors. PMID:26878228

  9. Neuroprotective properties of peroxisome proliferator-activated receptor alpha (PPARα) and its lipid ligands.

    PubMed

    Fidaleo, Marco; Fanelli, Francesca; Ceru, Maria Paola; Moreno, Sandra

    2014-01-01

    Signalling lipids are known to control a wide array of cellular processes, including cell proliferation, apoptosis, migration, and energy metabolism. Fatty acids and their derivatives, eicosanoids, phosphoinositides, sphingolipids, some cannabinoid-like molecules bind and activate nuclear receptors, including peroxisome proliferator-activated receptors (PPARs). This subfamily of transcription factors comprises three isotypes - PPARα (NR1C1), PPAR β/δ (NR1C2), PPARγ (NR1C3) - which bind to specific DNA response elements, as heterodimers with retinoid X receptors. PPAR activity is modulated by post-translational modifications and cofactors, towards which they show differential affinity. The three PPARs mutually interact, being integrated in a complex system, leading to the concept of a "PPAR triad". Nevertheless, the isotypes also show distinct actions on cellular physiology and partially different tissue, ligand and target gene specificities. In the brain, while the functions of PPARγ and its ligands are being thoroughly investigated, the actual and potential roles of PPARα and β/δ are far from being clarified. PPARα appears especially intriguing, since it is selectively expressed in certain brain areas and neuronal/glial populations, and modulates antioxidant responses, neurotransmission, neuroinflammation, neurogenesis, and glial cell proliferation/differentiation. This receptor and its endogenous ligands, including oleoylethanoloamide (OEA) and palmitoylethanolamide (PEA), are involved in physiological and pathological responses, such as satiety, memory consolidation, and modulation of pain perception. The protective role of PPARα agonists in neurodegenerative diseases and in neuropsychiatric disorders makes manipulation of this pathway highly attractive as therapeutic strategy for neuropathological conditions. In this review, we focus on the pleiotropic functions of PPARα and its lipid ligands in the nervous tissue, devoting special attention to

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

    SciTech Connect

    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.

  11. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  12. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  13. Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.

    PubMed

    Gomez-Diaz, Carolina; Reina, Jaime H; Cambillau, Christian; Benton, Richard

    2013-01-01

    Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is "conformationally activated" upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors. PMID:23637570

  14. New Ligand Binding Function of Human Cerberus and Role of Proteolytic Processing in Regulating Ligand-Receptor Interactions and Antagonist Activity.

    PubMed

    Aykul, Senem; Martinez-Hackert, Erik

    2016-02-13

    Cerberus is a key regulator of vertebrate embryogenesis. Its biological function has been studied extensively in frog and mouse embryos. Its ability to bind and antagonize the transforming growth factor-β (TGF-β) family ligand Nodal is well established. Strikingly, the molecular function of Cerberus remains poorly understood. The underlying reason is that Cerberus is a complex, multifunctional protein: It binds and inhibits multiple TGF-β family ligands, it may bind and inhibit some Wnt family members, and two different forms with distinct activities have been described. In addition, sequence homology between frog and mammalian Cerberus is low, suggesting that previous studies, which analyzed frog Cerberus function, may not accurately describe the function of mammalian Cerberus. We therefore undertook to determine the molecular activities of human Cerberus in TGF-β family signaling. Using purified proteins, surface plasmon resonance, and reporter gene assays, we discovered that human Cerberus bound and inhibited the TGF-β family ligands Activin B, BMP-6, and BMP-7, but not the frog Cerberus ligand BMP-2. Notably, full-length Cerberus successfully blocked ligand binding to type II receptors, but the short form was less effective. In addition, full-length Cerberus suppressed breast cancer cell migration but the short form did not. Thus, our findings expand the roles of Cerberus as TGF-β family signaling inhibitor, provide a molecular rationale for the function of the N-terminal region, and support the idea that Cerberus could have regulatory activities beyond direct inhibition of TGF-β family signaling. PMID:26802359

  15. Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes.

    PubMed

    Degn, Søren E; Kjaer, Troels R; Kidmose, Rune T; Jensen, Lisbeth; Hansen, Annette G; Tekin, Mustafa; Jensenius, Jens C; Andersen, Gregers R; Thiel, Steffen

    2014-09-16

    Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement. PMID:25197071

  16. Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

    PubMed Central

    Degn, Søren E.; Kjaer, Troels R.; Kidmose, Rune T.; Jensen, Lisbeth; Hansen, Annette G.; Tekin, Mustafa; Jensenius, Jens C.; Andersen, Gregers R.; Thiel, Steffen

    2014-01-01

    Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement. PMID:25197071

  17. A categorical structure-activity relationship analysis of GPR119 ligands

    PubMed Central

    Kumar, Pritesh; Carrasquer, Carl A.; Carter, Arren; Song, Zhao-Hui; Cunningham, Albert R.

    2016-01-01

    The categorical structure-activity relationship (cat-SAR) expert system has been successfully used in the analysis of chemical compounds that cause toxicity. Herein we describe the use of this fragment-based approach to model ligands for the G protein-coupled receptor 119 (GPR119). Using compounds that are known GPR119 agonists and compounds that we have confirmed experimentally that are not GPR119 agonists, four distinct cat-SAR models were developed. Using a leave-one out validation routine, the best GPR119 model had an overall concordance of 99 %, a sensitivity of 99 %, and a specificity of 100 %. Our findings from the in-depth fragment analysis of several known GPR119 agonists were consistent with previously reported GPR119 structure-activity relationship (SAR) analyses. Overall, while our results indicate that we have developed a highly predictive cat-SAR model that can be potentially used to rapidly screen for prospective GPR119 ligands the applicability domain must be taken into consideration. Moreover, our study demonstrates for the first time, that the cat-SAR expert system can be used to model G protein-coupled receptor ligands, many of which are important therapeutic agents. PMID:25401513

  18. Syntheses, crystal structures, anticancer activities of three reduce Schiff base ligand based transition metal complexes

    NASA Astrophysics Data System (ADS)

    Chang, Hui-Qin; Jia, Lei; Xu, Jun; Zhu, Tao-Feng; Xu, Zhou-Qing; Chen, Ru-Hua; Ma, Tie-Liang; Wang, Yuan; Wu, Wei-Na

    2016-02-01

    Three nickel(II) complexes, [Ni2(L1)2(tren)2(H2O)](ClO4)3 (1), [NiL2(tren)2](ClO4)·2.5H2O (2), [NiL2(tren)2]I·1.5H2O·CH3OH (3) based on amino acid reduced Schiff ligands are synthesized and characterized by physico-chemical and spectroscopic methods. The results show that in all complexes, the amino acid ligand is deprotonated and acts as an anionic ligand. In the dinuclear complex 1, each Ni(II) atom has a distorted octahedron geometry while with different coordination environment. However, the complexes 2 and 3 are mononuclear, almost with the same coordination environment. Furthermore, in vitro experiments are carried out, including MTT assay, Annexin V/PI flow cytometry and western blotting, to assess whether the complexes have antitumor effect. And the results show that all the three complexes have moderate anticancer activity towards human hepatic cancer (HepG2), human cervical cancer (HeLa) and human prostate (PC3) cell lines, in a concentration dependent way. The complex 1 exhibit higher cytotoxicity than the other two complexes and can induce human hepatic cancer cell (HepG2) to cell apoptosis by activating caspase 3.

  19. Cholinesterase inhibitory activity of chlorophenoxy derivatives-Histamine H3 receptor ligands.

    PubMed

    Łażewska, Dorota; Jończyk, Jakub; Bajda, Marek; Szałaj, Natalia; Więckowska, Anna; Panek, Dawid; Moore, Caitlin; Kuder, Kamil; Malawska, Barbara; Kieć-Kononowicz, Katarzyna

    2016-08-15

    In recent years, multitarget-directed ligands have become an interesting strategy in a search for a new treatment of Alzheimer's disease. Combination of both: a histamine H3 receptor antagonist/inverse agonist and a cholinesterases inhibitor in one molecule could provide a new therapeutic opportunity. Here, we present biological evaluation of histamine H3 receptor ligands-chlorophenoxyalkylamine derivatives against cholinesterases: acetyl- and butyrylcholinesterase. The target compounds showed cholinesterase inhibitory activity in a low micromolar range. The most potent in this group was 1-(7-(4-chlorophenoxy)heptyl)homopiperidine (18) inhibiting the both enzymes (EeAChE IC50=1.93μM and EqBuChE IC50=1.64μM). Molecular modeling studies were performed to explain the binding mode of 18 with histamine H3 receptor as well as with cholinesterases. PMID:27445168

  20. Simple ligand effects switch a hydrogenase mimic between H2 and O2 activation.

    PubMed

    Kim, Kyoungmok; Matsumoto, Takahiro; Robertson, Andrew; Nakai, Hidetaka; Ogo, Seiji

    2012-06-01

    Herein, we report a [NiRu] biomimetic system for O(2)-tolerant [NiFe]hydrogenases and demonstrate that electron donation to the [NiRu] center can switch the system between the activation of H(2) and O(2) through simple ligand effects by using hexamethylbenzene and pentamethylcyclopentadienyl ligands, respectively. Furthermore, we present the synthesis and direct observations of a [NiRu]-peroxo species, which was formed by the oxygenation of a Ni-SIa model [NiRu] complex, that we propose as a biomimetic analogue of O(2)-bound species (OBS) of O(2)-tolerant [NiFe]hydrogenases. The [NiRu]-peroxo complex was fully characterized by X-ray analysis, X-ray photoelectron spectroscopy (XPS), mass spectrometry, and (1)H NMR spectroscopy. The OBS analogue was capable of oxidizing p-hydroquinone and sodium borohydride to turn back into the Ni-SIa model complex. PMID:22383335

  1. Antiproliferative activity of ruthenium(ii) arene complexes with mono- and bidentate pyridine-based ligands.

    PubMed

    Richter, Stefan; Singh, Sushma; Draca, Dijana; Kate, Anup; Kumbhar, Anupa; Kumbhar, Avinash S; Maksimovic-Ivanic, Danijela; Mijatovic, Sanja; Lönnecke, Peter; Hey-Hawkins, Evamarie

    2016-08-16

    A series of Ru(II) arene complexes of mono- and bidentate N-donor ligands with carboxyl or ester groups and chlorido ancillary ligands were synthesised and structurally characterised. The complexes have a distorted tetrahedral piano-stool geometry. The binding interaction was studied with calf thymus DNA (CT-DNA) by absorption titration, viscosity measurement, thermal melting, circular dichroism, ethidium bromide displacement assay and DNA cleavage of plasmid DNA (pBR322), investigated by gel electrophoresis. The dichlorido complexes bind covalently to DNA in the dark, similar to cisplatin, while the monochlorido complexes bind covalently on irradiation, similar to cisplatin analogues. The compounds are selectively cytotoxic against several tumour cell lines and show specific nonlinear correlation between dose and activity. This phenomenon is closely related to their potential to act preferentially as inhibitors of cell division. PMID:27264161

  2. Ligand Controlled Morphology Evolution of Active Intermediates for the Syntheses of Gold Nanostars.

    PubMed

    Meng, Xianghua; Baride, Aravind; Jiang, Chaoyang

    2016-07-01

    Gold nanostars have unique plasmonic properties that are related to the highly branched nanostructures. However, it is challenging to precisely control these branches. Here we studied the reaction kinetics on the seed-mediated growth process of gold nanostars using in situ UV-vis spectroscopy. The impact of hydroquinone ligands on the formation and evolution of active intermediates was systematically explored. In addition, we improved the classical seed-mediated method to achieve a much better control on the final morphology of gold nanostars by a sudden addition of a high concentration ligand solution. Our method can significantly advance the syntheses of gold nanostars and provide numerous opportunities to prepare nanomaterials with unique morphology and plasmonic properties. PMID:27291864

  3. New structure-activity relationships of N-acetamide substituted pyrazolopyrimidines as pharmacological ligands of TSPO.

    PubMed

    Li, Jun; Schulte, Michael L; Nickels, Michael L; Manning, H Charles

    2016-08-01

    Translocator protein (TSPO) represents an attractive target for molecular imaging and therapy due to its prevalence and critical roles played in oncology and other pathologies. Based upon our previously optimized pyrazolopyrimidine scaffold, we elucidated new structure activity relationships related to N,N-disubstitutions of the terminal acetamide on pyrazolopyrimidines and further explored the impacts of these substituents on lipophilicity and plasma protein binding. Several novel chemical probes reported here exhibited significantly increased binding affinity, suitable lipophilicity and protein binding compared with contemporary TSPO ligands. We illustrate that N,N-acetamide disubstitution affords opportunities to introduce diverse chemical moieties distal to the central pyrazolopyrimidine core, without sacrificing TSPO affinity. We anticipate that further exploration of N-acetamide substitutions may yield additional TSPO ligands capable of furthering the field of precision medicine. PMID:27353534

  4. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  5. Synthesis, spectroscopic, coordination and biological activities of some organometallic complexes derived from thio-Schiff base ligands

    PubMed Central

    Abou-Hussein, Azza A.; Linert, Wolfgang

    2014-01-01

    Two series of mono- and binuclear complexes cyclic or acyclic thio-ferocine Schiff base ligands, derived from the condensation of 2-aminobenzenthiol (L) with monoacetyl ferrocene in the molar ratio 1:1 or in the molar ratio 1:2 for diacetyl ferocine have been prepared. The condensation reactions yield the corresponding Schiff Base ligands, HLa-Maf and H2Lb-Daf. The chelation of the ligands to metal ions occurs through the sulfur of the thiol group as well as the nitrogen atoms of the azomethine group of the ligands. HLa-Maf acts as monobasic bidentate or dibasic tetradentate, while H2Lb-Daf behaves as twice negatively cargend tetradentate ligand. The structures of these ligands were elucidated by elemental analysis, infrared, ultraviolet–visible spectra, as well as 1H NMR spectra. Reactions of the Schiff bases ligands with ruthenium(III), oxovanadium(IV) and dioxouranium(VI) afforded the corresponding transition metal complexes. The properties of the newly prepared complexes were analyse by elemental analyses, infrared, electronic spectra, 1H NMR as well as the magnetic susceptibility and conductivity measurement. The metal complexes exhibits different geometrical arrangements such as octahedral and square pyramidal coordination. Schiff base ligands and their metal complexes were tested against two pathogenic bacteria as Gram-positive and Gram-negative bacteria as well as one kind of fungi to study their biological activity. All the complexes exhibit antibacterial and antifungal activities against these organisms. PMID:24070648

  6. Synthesis, spectroscopic, coordination and biological activities of some organometallic complexes derived from thio-Schiff base ligands

    NASA Astrophysics Data System (ADS)

    Abou-Hussein, Azza A.; Linert, Wolfgang

    2014-01-01

    Two series of mono- and binuclear complexes cyclic or acyclic thio-ferocine Schiff base ligands, derived from the condensation of 2-aminobenzenthiol (L) with monoacetyl ferrocene in the molar ratio 1:1 or in the molar ratio 1:2 for diacetyl ferocine have been prepared. The condensation reactions yield the corresponding Schiff Base ligands, HLa-Maf and H2Lb-Daf. The chelation of the ligands to metal ions occurs through the sulfur of the thiol group as well as the nitrogen atoms of the azomethine group of the ligands. HLa-Maf acts as monobasic bidentate or dibasic tetradentate, while H2Lb-Daf behaves as twice negatively cargend tetradentate ligand. The structures of these ligands were elucidated by elemental analysis, infrared, ultraviolet-visible spectra, as well as 1H NMR spectra. Reactions of the Schiff bases ligands with ruthenium(III), oxovanadium(IV) and dioxouranium(VI) afforded the corresponding transition metal complexes. The properties of the newly prepared complexes were analyse by elemental analyses, infrared, electronic spectra, 1H NMR as well as the magnetic susceptibility and conductivity measurement. The metal complexes exhibits different geometrical arrangements such as octahedral and square pyramidal coordination. Schiff base ligands and their metal complexes were tested against two pathogenic bacteria as Gram-positive and Gram-negative bacteria as well as one kind of fungi to study their biological activity. All the complexes exhibit antibacterial and antifungal activities against these organisms.

  7. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding.

    PubMed

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  8. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

    PubMed Central

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H.; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  9. Ethanol inhibits ligand-activated calcium channels in human B lymphocytes

    SciTech Connect

    Mazer, B.D.; Brodie, C.; Domenico, J.; Gelfand, E.W. )

    1991-03-11

    The authors examined the acute effect of ethanol on the Ca{sup 2+} signal induced by two ligands, anti-{mu} antibody and platelet activating factor (PAF), during activation of human EBV-transformed B-cell lines. Stimulation of cells with either ligand results in an increase in free cytosolic Ca{sup 2+} as a result of release of Ca{sup 2+} from internal stores and of Ca{sup 2+} entry across the plasma membrane. Cells pretreated with 0.5% ethanol displayed a significantly smaller Ca{sup 2+} response following binding of either anti-{mu} or PAF, while ethanol itself did not effect basal levels of Ca{sup 2+}. The inhibitory effect of ethanol was time-dependent, the maximal effect being reached after 30 min of pre-incubation. In an attempt to define which component of the ligand-induced Ca{sup 2+} signal was inhibited by ethanol, the authors performed experiments in Ca{sup 2+}-free medium to eliminate Ca{sup 2+} influx. Ethanol treatment did not affect ligand-induced Ca{sup 2+} release from internal stores, but decreased the Ca{sup 2+} influx upon reintroduction of 1 mM Ca{sup 2+}. Ethanol also inhibited the Ca{sup 2+}-influx dependent induction of c-fos, triggered by anti-{mu} or PAF. The inhibitory effects of ethanol on Ca{sup 2+} channels in B cells are similar to those already reported in the CNS and may provide a basis for the immunosuppressive effect of ethanol.

  10. Small Molecule Receptor Protein Tyrosine Phosphatase γ (RPTPγ) Ligands That Inhibit Phosphatase Activity via Perturbation of the Tryptophan-Proline-Aspartate (WPD) Loop

    SciTech Connect

    Sheriff, Steven; Beno, Brett R; Zhai, Weixu; Kostich, Walter A; McDonnell, Patricia A; Kish, Kevin; Goldfarb, Valentina; Gao, Mian; Kiefer, Susan E; Yanchunas, Joseph; Huang, Yanling; Shi, Shuhao; Zhu, Shirong; Dzierba, Carolyn; Bronson, Joanne; Macor, John E; Appiah, Kingsley K; Westphal, Ryan S; O’Connell, Jonathan; Gerritz, Samuel W

    2012-11-09

    Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an 'open' conformation or a 'closed' conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced 'superopen' conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.

  11. Tumor Suppressor Activity of Profilin Requires a Functional Actin Binding Site

    PubMed Central

    Wittenmayer, Nina; Jandrig, Burkhard; Rothkegel, Martin; Schlüter, Kathrin; Arnold, Wolfgang; Haensch, Wolfgang; Scherneck, Siegfried; Jockusch, Brigitte M.

    2004-01-01

    Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1. PMID:14767055

  12. Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells

    PubMed Central

    Martin-Orozco, Natalia; Li, Yufeng; Wang, Yijun; Liu, Shijuan; Hwu, Patrick; Liu, Yong-Jun; Dong, Chen; Radvanyi, Laszlo

    2010-01-01

    CD4+CD25+Foxp3+ T-regulatory cells (Tregs) accumulate in tumors, however little is known about how the tumor environment influences this process. Here we show that human melanomas express ICOS-ligand (ICOS-L/B7H) that can provide costimulation through ICOS for the expansion of activated Tregs maintaining high Foxp3 and CD25 expression as well as suppressive function. Thus, ICOS-L expression by melanoma tumor cells may directly drive Treg activation and expansion in the tumor microenvironment as another mechanism of immune evasion. PMID:21098714

  13. Aspects of C-H Activation in Metal Complexes Containing Sulfur Ligands

    SciTech Connect

    Rakowski-DuBois, Mary C.

    2004-10-08

    The research project proposed to synthesize new metal complexes with sulfido, disulfido and other types of reactive sulfur ligands, and to explore the joint reactivity of metal and sulfur ligands with hydrogen and organic molecules. The overall objective was to investigate reaction pathways relevant to those observed for the heterogeneous metal sulfide catalysts which promote hydrogen activation, hydrogenation-dehydrogenation of organic substrates, and hydrogenolysis of carbon-heteroatom bonds. Particular emphasis was placed on CpRe derivatives (where Cp might be C5H5 or alkylated versions) so that comparisons could be made with the previously studied CpMo complexes, which showed extensive reactivity at the sulfur ligands. Heterogeneous rhenium sulfides generally show higher catalytic activity than molybdenum sulfides, and this is attributed, in part, to the weaker Re-S bond strength, relative to the moybdenum-sulfur bond. In our studies of discrete Re-sulfide complexes, we have also observed evidence for weaker Re-S bonds relative to the molybdenum systems. In addition we have characterized novel hydrogen activation by rhenium sulfido complexes, as well as carbon-hydrogen, carbon-sulfur and metal sulfur bond cleavage reactions. Hydrogen Activation. The complex Cp{prime}ReCl2S3 was synthesized in ca 70% yield and characterized by an X-ray diffraction study which confirms that the complex contains a {eta}2-trisulfide ligand. The cyclic voltammogram of Cp{prime}ReCl2S3 shows a wide window of redox stability with an irreversible reduction wave at -0.97 V and an irreversible oxidation at +1.03 V vs Fc. Nevertheless, the complex undergoes a facile reaction with hydrogen at 50 C to form H2S and a new dinuclear sulfido bridged rhenium complex. This reaction is of interest because it is the first example of the hydrogenolysis of a discrete metal polysulfide complex to produce H2S, a reaction also observed for heterogeneous rhenium sulfides. The reaction contrasts with

  14. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites.

    PubMed

    McCullough, Christopher R; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy; Sem, Daniel S

    2009-03-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a (13)CH(3)-reporter attached. This (13)C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  15. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

    PubMed

    Wang, Chunhe; Mooney, Jeffery L; Meza-Romero, Roberto; Chou, Yuan K; Huan, Jianya; Vandenbark, Arthur A; Offner, Halina; Burrows, Gregory G

    2003-08-15

    Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells. PMID:12902496

  16. Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.

    PubMed

    Chen, G; Radominska-Pandya, A

    2000-10-17

    Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution. PMID:11027136

  17. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  18. Dynamics of the Ligand Binding Domain Layer during AMPA Receptor Activation.

    PubMed

    Baranovic, Jelena; Chebli, Miriam; Salazar, Hector; Carbone, Anna L; Faelber, Katja; Lau, Albert Y; Daumke, Oliver; Plested, Andrew J R

    2016-02-23

    Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample. PMID:26910426

  19. Antispermatogenic Activity of the Benzothiazoline Ligand and Corresponding Organoantimony(V) Derivative in Male Albino Rats

    PubMed Central

    Sharma, Pankaj K.; Rehwani, H.; Rai, A. K.; Gupta, R. S.; Singh, Y. P.

    2006-01-01

    Triphenylantimony(V) derivative, Ph3Sb(OPri) [SC6H4N : C(CH3)CH2C(O)CH3], 1b, and the corresponding benzothiazoline ligand [1, 2], HNC6H4SC⎴(CH3)CH2C(O)CH3, 1a, have been tested for their effects on the reproductive system of male albino rats. The oral administration of both 1a and 1b at the dose level of 10 mg/rat/day produced significant reduction in the weights of testes, epididymides, seminal vesicles, and ventral prostate. Significant decrease in sperm motility as well as in sperm density resulted in 100% sterility. Significant (P < .01) alterations were also found in biochemical parameters of reproductive organs in treated male rats as compared to the control group. Production of preleptotene, pachytene, and secondary spermatocytes was decreased by 42%, 43%, 39%, and by 44%, 49%, 55% in the ligand, 1a, and organoantimony(V) derivative, 1b, treated rats, respectively. These results indicate that both compounds 1a and 1b are antispermatogenic in nature and on oral administration in male rats, and finally caused sterility. A comparison indicates that the organoantimony(V) derivative 1b is more effective pertaining to its antispermatogenic activity than the corresponding ligand 1a. PMID:17496999

  20. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  1. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  2. Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.

    PubMed

    Glasgow, B J; Gasymov, O K; Abduragimov, A R; Yusifov, T N; Altenbach, C; Hubbell, W L

    1999-10-12

    Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the

  3. Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications

    PubMed Central

    2014-01-01

    Background A useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system. Results A convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells. Conclusions A new, efficient cultivation system for enhanced secretory

  4. Receptor Tyrosine Kinases, TYRO3, AXL, and MER, Demonstrate Distinct Patterns and Complex Regulation of Ligand-induced Activation*

    PubMed Central

    Tsou, Wen-I; Nguyen, Khanh-Quynh N.; Calarese, Daniel A.; Garforth, Scott J.; Antes, Anita L.; Smirnov, Sergey V.; Almo, Steve C.; Birge, Raymond B.; Kotenko, Sergei V.

    2014-01-01

    TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs. PMID:25074926

  5. Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R).

    PubMed

    Morton, H Craig; Pleass, Richard J; Woof, Jenny M; Brandtzaeg, Per

    2004-12-24

    Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding. PMID:15485844

  6. Structural Diversity of Cadmium(II) Coordination Polymers Induced by Tuning the Coordination Sites of Isomeric Ligands.

    PubMed

    Liu, Bo; Zhou, Hui-Fang; Hou, Lei; Wang, Jian-Ping; Wang, Yao-Yu; Zhu, Zhonghua

    2016-09-01

    When the coordination sites of ligands were shifted, the solvothermal reactions of four positional isomeric asymmetrical pyridyldicarboxylatic acids with Cd(NO3)2 generated four new coordination polymers, [Cd(L1)(DMF)3]·DMF·H2O (1), [H2N(CH3)2]2[Cd(L2)2]·3DMF·H2O (2), [Cd(L3)(H2O)2] (3), and [Cd(L4)]·1.5DMF (4), where DMF = N,N-dimethylformamide, H2L1 = 2-(3'-carboxylphenyl)isonicotinic acid, H2L2 = 2-(4'-carboxylphenyl)isonicotinic acid, H2L3 = 5-(3'-carboxylphenyl)nicotic acid, and H2L4 = 2-(3'-pyridyl)terephthalic acid. 1 shows a rare 2D fabric structure. 2 discloses a grid-layer structure with heterochiral helical chains and in which three sets of layers stack in different directions, affording an unprecedented 2D + 2D + 2D → 3D polycatenating framework with 3D intersecting porous systems. 3 also displays a 2D layer possessing strong intralayer π···π interactions and interlayer hydrogen bonds. 4 contains a rare Cd2(COO)4 paddle-wheel unit and forms a 3D framework with 1D open channels. The carboxyl and pyridyl groups of the positional isomeric H2L1-H2L4 ligands show distinct bridging fashions, which leads to the production of versatile architectures of 1-4, and their effects on the crystal structures are discussed. 1-4 reveal solid-state photoluminescence stemming from intraligand charge transfer. 2 and 4 show high selectivity for CO2 over CH4 but with different CO2 adsorption enthalpies. Grand canonical Monte Carlo simulations identified the multiple adsorption sites in 2 for CO2. PMID:27513092

  7. Interaction of cucurbitacins with human serum albumin: Thermodynamic characteristics and influence on the binding of site specific ligands.

    PubMed

    Abou-Khalil, Rony; Jraij, Alia; Magdalou, Jacques; Ouaini, Naïm; Tome, Daniel; Greige-Gerges, Hélène

    2009-06-01

    Cucurbitacins (Cuc) are cytotoxic oxygenated triterpenes. Their binding to albumin may control their diffusion and consequently their biological effects. The specific binding site of Cuc to albumin is important to be defined as it could determine some of the drug interactions of the compounds. This paper deals with the interaction between human serum albumin and a series of four cucurbitacins (B, D, E and I) measured by fluorescence and circular dichroism spectroscopies. Cuc B and E at C25, are the acetylated forms of Cuc D and I. The binding parameters (K(a) and n) of Cuc B, D and E to albumin were determined at 288, 293, 298 and 303K. Cuc B possesses the higher binding constant (K(a)) values followed by Cuc E and D. The thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. They indicated hydrophobic and electrostatic interactions for Cuc B, hydrophobic interaction for Cuc E, hydrophobic and hydrogen bond interactions for Cuc D. In addition to bilirubin, Cuc B, D, and E increased the binding constant values for warfarin to albumin, whereas they did not affect the binding of other ligands of site I such as chloroform and salicylate. The increase of the K(a) values of warfarin and bilirubin was associated with an increase of the binding constant value of cucurbitacin to albumin. Cuc I did not bind to albumin and could be considered less capable to affect the interaction of ligands to albumin than Cuc B, D and E. CD spectra indicated that Cuc binding to HSA was not associated with substantial structural changes of the protein. PMID:19380237

  8. Dual ligand/receptor interactions activate urothelial defenses against uropathogenic E. coli

    PubMed Central

    Liu, Yan; Mémet, Sylvie; Saban, Ricardo; Kong, Xiangpeng; Aprikian, Pavel; Sokurenko, Evgeni; Sun, Tung-Tien; Wu, Xue-Ru

    2015-01-01

    During urinary tract infection (UTI), the second most common bacterial infection, dynamic interactions take place between uropathogenic E. coli (UPEC) and host urothelial cells. While significant strides have been made in the identification of the virulence factors of UPEC, our understanding of how the urothelial cells mobilize innate defenses against the invading UPEC remains rudimentary. Here we show that mouse urothelium responds to the adhesion of type 1-fimbriated UPEC by rapidly activating the canonical NF-κB selectively in terminally differentiated, superficial (umbrella) cells. This activation depends on a dual ligand/receptor system, one between FimH adhesin and uroplakin Ia and another between lipopolysaccharide and Toll-like receptor 4. When activated, all the nuclei (up to 11) of a multinucleated umbrella cell are affected, leading to significant amplification of proinflammatory signals. Intermediate and basal cells of the urothelium undergo NF-κB activation only if the umbrella cells are detached or if the UPEC persistently express type 1-fimbriae. Inhibition of NF-κB prevents the urothelium from clearing the intracellular bacterial communities, leading to prolonged bladder colonization by UPEC. Based on these data, we propose a model of dual ligand/receptor system in innate urothelial defenses against UPEC. PMID:26549759

  9. Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8

    PubMed Central

    Helmke, Christina; Raab, Monika; Rödel, Franz; Matthess, Yves; Oellerich, Thomas; Mandal, Ranadip; Sanhaji, Mourad; Urlaub, Henning; Rödel, Claus; Becker, Sven; Strebhardt, Klaus

    2016-01-01

    Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression. PMID:27325299

  10. A study of in vitro antibacterial activity of lanthanides complexes with a tetradentate Schiff base ligand

    PubMed Central

    Al Momani, Waleed Mahmoud; Taha, Ziyad Ahmed; Ajlouni, Abdulaziz Mahmoud; Shaqra, Qasem Mohammad Abu; Al Zouby, Muaz

    2013-01-01

    Objective To establish the antibacterial activity of lanthanides complexes with a tetradentate Schiff base ligand L. Methods (N, N′-bis (1-naphthaldimine)-o-phenylenediamine) was prepared from the condensation of 2-hydroxy-1-naphthaldehyde with o-phenylenediamine in a molar ratio of 2:1. The antimicrobial activity of the resultant Ln (III) complexes was investigated using agar well diffusion and micro-broth dilution techniques; the latter was used to establish the minimum inhibitory concentrations for each compound investigated. Results Most of Ln (III) complexes were found to exhibit antibacterial activities against a number of pathogenic bacteria with MICs ranging between 1.95-250.00 µg/mL. Staphylococcus aureus was the most susceptible bacterial species to [LaL(NO3)2(H2O)](NO3) complex while Shigella dysenteriae and Escherichia coli required a relatively higher MIC (250 µg/mL). The complexes La (III) and Pr (III) were effective inhibitors against Staphylococcus aureus, whereas Sm (III) complex was effective against Serratia marcescens. On the other hand, Gd (III), La (III) and Nd (III) were found to be more potent inhibitors against Pseudomonas aeruginosa than two of commonly used antibiotics. The remaining Ln (III) complexes showed no remarkable activity as compared to the two standard drugs used. Conclusions Tetradentate Schiff base ligand L and its complexes could be a potential antibacterial compounds after further investigation. PMID:23646299

  11. Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8.

    PubMed

    Helmke, Christina; Raab, Monika; Rödel, Franz; Matthess, Yves; Oellerich, Thomas; Mandal, Ranadip; Sanhaji, Mourad; Urlaub, Henning; Rödel, Claus; Becker, Sven; Strebhardt, Klaus

    2016-08-01

    Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression. PMID:27325299

  12. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand. PMID:25916672

  13. Differential Effects of Methoxy Group on the Interaction of Curcuminoids with Two Major Ligand Binding Sites of Human Serum Albumin

    PubMed Central

    Sato, Hiroki; Chuang, Victor Tuan Giam; Yamasaki, Keishi; Yamaotsu, Noriyuki; Watanabe, Hiroshi; Nagumo, Kohei; Anraku, Makoto; Kadowaki, Daisuke; Ishima, Yu; Hirono, Shuichi; Otagiri, Masaki; Maruyama, Toru

    2014-01-01

    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. PMID:24498401

  14. Alteration of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.

    PubMed

    Le Gros, G; Zhang, X M; Parsons, P G

    1994-12-01

    Nontoxic doses of the histamine H2 antagonists ranitidine, cimetidine, lamtidine and mifentidine rapidly and reversibly increased tyrosinase activity in an amelanotic human melanoma cell line (MM96L) with low constitutive activity. The H2 antagonists, famotidine and MGTI, and the imidazol(in)e receptor ligand clonidine had no effect either alone or in competition with ranitidine, whilst metiamide decreased tyrosinase activity. Lysosomotropic amines had a similar effect to ranitidine, except that induction reached a plateau at 6 h and was insensitive to amiloride. Human melanocytes and pigmented human melanoma cell lines exhibited minimal levels of tyrosinase induction, which was dependent on protein synthesis but not on RNA or DNA synthesis. Constitutive tyrosinase activity in MM96L cells was much less stable than in melanocytes and pigmented melanoma cells. No change was observed in expression of gp75, neural specific octamer binding proteins, or in mRNA levels of tyrosinase, Pmel-17 and gp75 (TRP-1). Tyrosinase was inhibited by the H3 agonist imetit but not by alpha-methylhistamine or the H3 antagonist thioperamide. Overall, this work showed that certain H2 antagonists activate an unstable form of tyrosinase in amelanotic melanoma cells by a post-transcriptional mechanism dependent on protein synthesis. An imidazoline/guanidinium receptor site rather than the H2 receptor appeared to be involved. PMID:7535606

  15. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms

    PubMed Central

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F.; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems

  16. The influence of ligand-activated LXR on primary human trophoblasts

    PubMed Central

    Larkin, Jacob C.; Sears, Sarah B.; Sadovsky, Yoel

    2014-01-01

    Introduction The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. Methods We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. Results Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. Conclusion These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands. PMID:25255963

  17. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms.

    PubMed

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires' disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in

  18. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

    SciTech Connect

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao; Han, Xiang Hua; Lee, Dong-Ho; Lee, Hak-Ju; Hwang, Bang Yeon; Lee, Sung-Joon

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  19. Optically active P5-deltacyclenes: selective oxidation, ligand properties, and a diastereoselective rearrangement reaction.

    PubMed

    Keller, I C; Bauer, W; Heinemann, F W; Höhn, C; Rohwer, L; Zenneck, U

    2016-04-25

    Cage-chiral tetra-tert-butyl-P5-deltacyclene is accessible as a pair of highly enriched enantiomers and . The only secondary phosphorus atom P1 of the cage can be selectively oxidized by reaction with t-BuOOH. The P1-oxo species and , allow the direct determination of their ee values. Oxidation occurs with the complete retention of the optical activity of the compounds. The chiroptical properties of and are strongly dominated by their cage chirality, the oxygen atom does not contribute significantly. Elemental sulfur and selenium oxidize P5 with high preference to yield P5-thio- and P5-seleno-P5-deltacyclenes and of the intact cages again. Longer reaction time and more than stoichiometric amounts of selenium, leads to tri-seleno-P5-tetracycloundecane , a partially opened oxidized rearrangement product. The ligand properties of racemic were determined. Diphosphetane phosphorus atom P2 of is the active donor center to bind a Cr(CO)5 fragment, but a tautomerization of takes place if [(benzene)RuCl2]2 is added. A hydrogen atom migrates from P1 to the oxygen atom to form a phosphinous acid ligand. The lone pair of P1 is regenerated and acts as the active ligand function of the cage in this case. As for , the base n-BuLi induces an efficient cage rearrangement reaction of , where P1 and the neighboring carbon atom C4 containing its t-Bu substituent change places. C4 moves to its new position without breaking the bond with P5, this way forming the novel P1-oxo-P5-norsnoutene cage in a highly diastereoselective process. PMID:27055252

  20. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  1. IDENTIFICATION OF THE GPR55 ANTAGONIST BINDING SITE USING A NOVEL SET OF HIGH POTENCY GPR55 SELECTIVE LIGANDS

    PubMed Central

    Kotsikorou, Evangelia; Sharir, Haleli; Shore, Derek M.; Hurst, Dow P.; Lynch, Diane L.; Madrigal, Karla E.; Heynen-Genel, Susanne; Milan, Loribelle B.; Chung, Thomas D.Y.; Seltzman, Herbert H.; Bai, Yushi; Caron, Marc G.; Barak, Larry S.; Croatt, Mitchell P.; Abood, Mary E.; Reggio, Patricia H.

    2014-01-01

    GPR55 is a Class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as L-α-lysophosphatidylinositol (LPI). While there is increasing evidence for physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300,000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50s in the 0.16 to 2.72 μM range (Heynen-Genel, et al., (2010) “Screening for Selective Ligands for GPR55 – Antagonists” [ML191, ML192, ML193] Bookshelf ID: NBK66153; PMID: 22091481). Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1 or CB2 up to 20 μM. Using a model of GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second generation GPR55 selective antagonists. PMID:24274581

  2. Characterization of a second ligand binding site of the insulin receptor

    SciTech Connect

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan . E-mail: jonathan.whittaker@case.edu

    2006-08-18

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the {alpha} subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K {sub d} of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.

  3. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  4. Stereochemical influences upon the opioid ligand activities of 4-alkyl-4-arylpiperidine derivatives.

    PubMed

    Casy, A F; Dewar, G H; al Deeb, O A

    1989-01-01

    The synthesis and stereochemistry (configuration and preferred solute conformation) of some 4-alkyl (methyl, n-propyl, isobutyl)-4-(3-hydroxy-phenyl)-1-methylpiperidines and corresponding 3-methyl diastereoisomeric pairs are reported, together with their in vivo and in vitro activities as opioid ligands. All potent agonists exhibit a preference for axial 4-aryl chair conformations when protonated, and stereochemical analogies with rigid opioids of the benzomorphan class are discussed. Antagonist properties are found in compounds with preference for equatorial 4-aryl chairs, notably the cis 3,4-dimethyl derivative. PMID:2561991

  5. Spatiotemporal Regulation of Hsp90–Ligand Complex Leads to Immune Activation

    PubMed Central

    Tamura, Yasuaki; Yoneda, Akihiro; Takei, Norio; Sawada, Kaori

    2016-01-01

    Although heat shock proteins (HSPs) primarily play a pivotal role in the maintenance of cellular homeostasis while reducing extracellular as well as intracellular stresses, their role in immunologically relevant scenarios, including activation of innate immunity as danger signals, antitumor immunity, and autoimmune diseases, is now gaining much attention. The most prominent feature of HSPs is that they function both in their own and as an HSP–ligand complex. We here show as a unique feature of extracellular HSPs that they target chaperoned molecules into a particular endosomal compartment of dendritic cells, thereby inducing innate and adaptive immune responses via spatiotemporal regulation. PMID:27252703

  6. Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    SciTech Connect

    Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W. )

    1991-05-01

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

  7. Antimicrobial Activity of Metal & Metal Oxide Nanoparticles Interfaced With Ligand Complexes Of 8-Hydroxyquinoline And α-Amino Acids

    NASA Astrophysics Data System (ADS)

    Bhanjana, Gaurav; Kumar, Neeraj; Thakur, Rajesh; Dilbaghi, Neeraj; Kumar, Sandeep

    2011-12-01

    Antimicrobial nanotechnology is a recent addition to the fight against disease causing organisms, replacing heavy metals and toxins. In the present work, mixed ligand complexes of metals like zinc, silver etc. and metal oxide have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and N-and/O-donor amino acids such as L-serine, L-alanine, glycine, cysteine and histidine as secondary ligands. These complexes were characterized using different spectroscopic techniques. The complexes were tested for antifungal and antibacterial activity by using agar well diffusion bioassay.

  8. Isolation of pharmacologically active benzodiazepine receptor ligands from Tilia tomentosa (Tiliaceae).

    PubMed

    Viola, H; Wolfman, C; Levi de Stein, M; Wasowski, C; Peña, C; Medina, J H; Paladini, A C

    1994-08-01

    Tilia species are traditional medicinal plants widely used in Latin America as sedatives and tranquilizers. For this purpose, the infusion of their inflorescences is used to prepare a tea. In this study extracts of inflorescences from Tilia tomentosa Moench, one of the species found in the market, were purified using a benzodiazepine (BZD) binding assay to detect BZD receptor ligands in the different fractions. One of the ligands was identified as kaempferol, but it had low affinity (Ki = 93 microM) for this receptor, and did not produce sedative or anxiolytic effects in mice. On the other hand, a complex fraction, containing as yet unidentified constituents, but probably of a flavonoid nature, when administered intraperitoneally in mice, had a clear anxiolytic effect in both the elevated plus-maze and holeboard tests, two well validated pharmacological tests to measure anxiolytic and sedative compounds. This active fraction had no effect on total and ambulatory locomotor activity. In conclusion, our results demonstrate the occurrence of active principle(s) in, at least, one species of Tilia that may explain its ethnopharmacological use as an anxiolytic. PMID:7990504

  9. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  10. Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

    PubMed

    Newton, S M; Allen, J S; Cao, Z; Qi, Z; Jiang, X; Sprencel, C; Igo, J D; Foster, S B; Payne, M A; Klebba, P E

    1997-04-29

    Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane. PMID:9114029

  11. Synthesis and biological activity of small peptides as NOP and opioid receptors' ligands: view on current developments.

    PubMed

    Naydenova, Emilia; Todorov, Petar; Zamfirova, Rositza

    2015-01-01

    The heptadecapeptide nociceptin, also called orphanin FQ (N/OFQ), is the endogenous agonist of the N/OFQ peptide receptor (NOP receptor) and is involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents overview of the several recently reported NOP ligands (agonists and antagonists), with an emphasis of the structural features that may be important for modulating the intrinsic activity of these ligands. In addition, a brief account on the characterization of newly synthesized ligands of NOP receptor with aminophosphonate moiety and β-tryptophan analogues will be presented. PMID:25677770

  12. Charge is Major Determinant of Activation of the Ligand-Responsive Multidrug Resistance Gene Regulator, BmrR.

    PubMed

    Bachas, Sharrol; Kohrs, Bryan; Wade, Herschel

    2016-05-19

    A medium-throughput approach (80+ compounds) to investigate allosteric transcriptional control in the multidrug resistance gene regulator BmrR, with cations, zwitterions, uncharged compounds and anions, is described. Even at the allosteric level, BmrR is quite promiscuous with regard to molecular shape and structure, but it is sensitive to molecular charge. A role for charge is further supported by differences in the activation properties of structurally similar ligands displaying variable charge properties as well as differences in activation by zwitterions and uncharged ligands, which show similar binding affinities. A comparison of allosteric selectivity with the distribution of differently charged ligands in bacterial cellular environments suggests that the selectivity of charge is a major factor in discrimination of xenobiotics, and native biological compounds and metabolites. Interestingly, in eukaryotic cells, the selectivity of cationic ligands might be a protective mechanism against chemical agents that act in a promiscuous fashion. PMID:27010425

  13. A Series of Diamagnetic Pyridine Monoimine Rhenium Complexes with Different Degrees of Metal-to-Ligand Charge Transfer: Correlating (13) C NMR Chemical Shifts with Bond Lengths in Redox-Active Ligands.

    PubMed

    Sieh, Daniel; Kubiak, Clifford P

    2016-07-18

    A set of pyridine monoimine (PMI) rhenium(I) tricarbonyl chlorido complexes with substituents of different steric and electronic properties was synthesized and fully characterized. Spectroscopic (NMR and IR) and single-crystal X-ray diffraction analyses of these complexes showed that the redox-active PMI ligands are neutral and that the overall electronic structure is little affected by the choices of the substituent at the ligand backbone. One- and two-electron reduction products were prepared from selected starting compounds and could also be characterized by multiple spectroscopic methods and X-ray diffraction. The final product of a one-electron reduction in THF is a diamagnetic metal-metal-bonded dimer after loss of the chlorido ligand. Bond lengths in and NMR chemical shifts of the PMI ligand backbone indicate partial electron transfer to the ligand. Two-electron reduction in THF also leads to the loss of the chlorido ligand and a pentacoordinate complex is obtained. The comparison with reported bond lengths and (13) C NMR chemical shifts of doubly reduced free pyridine monoaldimine ligands indicates that both redox equivalents in the doubly reduced rhenium complex investigated here are located in the PMI ligand. With diamagnetic complexes varying over three formal reduction stages at the PMI ligand we were, for the first time, able to establish correlations of the (13) C NMR chemical shifts with the relevant bond lengths in redox-active ligands over a full redox series. PMID:27319753

  14. Computational approaches to identifying and characterizing protein binding sites for ligand design.

    PubMed

    Henrich, Stefan; Salo-Ahen, Outi M H; Huang, Bingding; Rippmann, Friedrich F; Cruciani, Gabriele; Wade, Rebecca C

    2010-01-01

    Given the three-dimensional structure of a protein, how can one find the sites where other molecules might bind to it? Do these sites have the properties necessary for high affinity binding? Is this protein a suitable target for drug design? Here, we discuss recent developments in computational methods to address these and related questions. Geometric methods to identify pockets on protein surfaces have been developed over many years but, with new algorithms, their performance is still improving. Simulation methods show promise in accounting for protein conformational variability to identify transient pockets but lack the ease of use of many of the (rigid) shape-based tools. Sequence and structure comparison approaches are benefiting from the constantly increasing size of sequence and structure databases. Energetic methods can aid identification and characterization of binding pockets, and have undergone recent improvements in the treatment of solvation and hydrophobicity. The "druggability" of a binding site is still difficult to predict with an automated procedure. The methodologies available for this purpose range from simple shape and hydrophobicity scores to computationally demanding free energy simulations. PMID:19746440

  15. Propionic acid secreted from propionibacteria induces NKG2D ligand expression on human-activated T lymphocytes and cancer cells.

    PubMed

    Andresen, Lars; Hansen, Karen Aagaard; Jensen, Helle; Pedersen, Stine Falsig; Stougaard, Peter; Hansen, Helle Rüsz; Jurlander, Jesper; Skov, Søren

    2009-07-15

    We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could directly stimulate functional MICA/B surface expression and MICA promoter activity by a mechanism dependent on intracellular calcium. Deletion and point mutations further demonstrated that a GC-box motif around -110 from the MICA transcription start site is essential for propionate-mediated MICA promoter activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate and CMV. In contrast, compounds with histone deacetylase-inhibitory activity such as butyrate and FR901228 stimulated MICA/B expression through a pathway that was not affected by inhibition of glycolysis, clearly suggesting that MICA/B is regulated through different molecular mechanisms. We propose that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic. PMID:19553547

  16. Including Ligand Induced Protein Flexibility into Protein Tunnel Prediction

    PubMed Central

    Kingsley, Laura J.; Lill, Markus A.

    2014-01-01

    In proteins with buried active sites, understanding how ligands migrate through the tunnels that connect the exterior of the protein to the active site can shed light on substrate specificity and enzyme function. A growing body of evidence highlights the importance of protein flexibility in the binding site upon ligand binding; however, the influence of protein flexibility throughout the body of the protein during ligand entry and egress is much less characterized. We have developed a novel tunnel prediction and evaluation method named IterTunnel, which includes the influence of ligand-induced protein flexibility, guarantees ligand egress, and provides detailed free energy information as the ligand proceeds along the egress route. IterTunnel combines geometric tunnel prediction with steered MD in an iterative process to identify tunnels that open as a result of ligand migration and calculates the potential of mean force (PMF) of ligand egress through a given tunnel. Applying this new method to cytochrome P450 2B6 (CYP2B6), we demonstrate the influence of protein flexibility on the shape and accessibility of tunnels. More importantly, we demonstrate that the ligand itself, while traversing through a tunnel, can reshape tunnels due to its interaction with the protein. This process results in the exposure of new tunnels and the closure of pre-existing tunnels as the ligand migrates from the active site. PMID:25043499

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

    PubMed

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

    2015-10-01

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

  18. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-01

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ‡G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  19. Mixed Ligand Complexes of N-Methyl-N-phenyl Dithiocarbamate: Synthesis, Characterisation, Antifungal Activity, and Solvent Extraction Studies of the Ligand

    PubMed Central

    Ekennia, Anthony C.; Onwudiwe, Damian C.; Ume, Cyril; Ebenso, Eno E.

    2015-01-01

    A series of mixed ligand dithiocarbamate complexes with a general formula [ML2(py)2], where M = Mn(II), Co(II), Ni(II), and Cu(II), py = pyridine, and L = N-methyl-N-phenyl dithiocarbamate have been prepared and characterised by elemental analysis, FTIR and Uv spectroscopy, magnetic moment, and thermogravimetric and conductance analysis. The infrared spectra showed that symmetrical bidentate coordination occurred with the dithiocarbamate moiety through the sulfur atoms, while neutral monodentate coordination occurred through the nitrogen atom for the pyridine molecule in the complexes. The electronic spectra, elemental analysis, and magnetic moment results proved that the complexes adopted octahedral geometry. The conductance measurement showed that the complexes are nonelectrolytes proving their nonionic nature. The compounds were screened for three human pathogenic fungi: Aspergillus flavus, Aspergillus niger, and Candida albicans. The cobalt complex showed the best antifungal activity among the test compounds. Liquid-liquid extractive abilities of the ligand towards copper and nickel ions in different solvent media were investigated. The ligand showed a strong binding affinity towards the metals ions with an extractive efficiency of about 99%. PMID:26543441

  20. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  1. New metal complexes of N3 tridentate ligand: Synthesis, spectral studies and biological activity

    NASA Astrophysics Data System (ADS)

    Al-Hamdani, Abbas Ali Salih; Al Zoubi, Wail

    2015-02-01

    New tridentate ligand 3-amino-4-{1,5-dimethyl-3-[2-(5-methyl-1H-indol-3-yl)-ethylimino]-2phenyl-2,3-dihydro-1H-pyrazol-4-ylazo}-phenol L was synthesized from the reaction of 1,5-dimethyl-3-[2-(5-methyl-1H-indol-3-yl)-ethylimino]-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylamine and 3.4-amino phenol. A complexes of these ligand [Ni(II)(L)(H2O)2 Cl]Cl, [pt(IV)(L)Cl3]Cl and [M(II)(L)Cl]Cl (M = Pd (II), Zn (II), Cd (II) and Hg (II) were synthesized. The complexes were characterized by spectroscopic methods and magnetic moment measurements, elemental analysis, metal content, Chloride containing and conductance. These studies revealed octahedral geometries for the Ni (II), pt (IV) complexes, square planar for Pd (II) complex and tetrahedral for the Zn (II), Cd(II) and Hg (II) complexes. The study of complexes formation via molar ratio and job method in DMF solution has been investigated and results were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1). The thermodynamic parameters, such as ΔE*, ΔH*, ΔS* ΔG* and K are calculated from the TGA curve using Coats-Redfern method. Hyper Chem-8 program has been used to predict structural geometries of compounds in gas phase. The synthesized ligand and its metal complexes were screened for their biological activity against bacterial species, two Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram negative bacteria (Escherichia coli and Pseudomonasaeruginosa).

  2. New RuII Complex for Dual Activity: Photoinduced Ligand Release and 1O2 Production

    PubMed Central

    Loftus, Lauren M.; White, Jessica K.; Albani, Bryan A.; Kohler, Lars; Kodanko, Jeremy J.; Thummel, Randolph P.

    2016-01-01

    The new complex [Ru(pydppn)(biq)(py)]2+ (1) undergoes both py photodissociation in CH3CN with Φ500=0.0070(4) and 1O2 production with ΦΔ=0.75(7) in CH3OH from a long-lived 3ππ* state centered on the pydppn ligand (pydppn=3-(pyrid-2-yl)benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine; biq = 2,2′-biquinoline; py= pyridine). This represents an order of magnitude decrease in the Φ500 compared to the previously reported model compound [Ru(tpy)(biq)(py)]2+ (3) (tpy=2,2′:6′,2″-terpyridine) that undergoes only ligand exchange. The effect on the quantum yields by the addition of a second deactivation pathway through the low-lying 3ππ* state necessary for dual reactivity was investigated using ultrafast and nanosecond transient absorption spectroscopy, revealing a significantly shorter 3MLCT lifetime in 1 relative to that of the model complex 3. Due to the structural similarities between the two compounds, the lower values of Φ500 and ΦΔ compared to that of [Ru(pydppn)(bpy)(py)]2+ (2) (bpy=2,2′-bipyridine) are attributed to a competitive excited state population between the 3LF states involved in ligand dissociation and the long-lived 3ππ* state in 1. Complex 1 represents a model compound for dual activity that may be applied to photochemotherapy. PMID:26715085

  3. Controlling the Growth and Catalytic Activity of Platinum Nanoparticles Using Peptide and Polymer Ligands

    NASA Astrophysics Data System (ADS)

    Forbes, Lauren Marie

    Heterogeneous catalysts have widespread industrial applications. Platinum nanomaterials in particular, due to their particularly high electrocatalytic activity and durability, are used to catalyze a wide variety of reactions, including oxygen reduction, which is frequently used as the cathode reaction in fuel cells. As platinum is a very expensive material, a high priority in fuel cell research is the exploration of less expensive, more efficient catalysts for the oxygen reduction reaction (ORR). We demonstrate here the use of phage display to identify peptides that bind to Pt (100) which were then used to synthesize platinum cubes in solution. However, while the peptides were able to control particle growth, the bio-synthesized Pt particles showed extremely poor activity when tested for ORR. This could be attributed to peptide coverage on the surface or strong interactions between particular amino acids and the metal that are detrimental for catalysis. To investigate this further, we decided to investigate the role of individual amino acids on Pt nanocrystal synthesis and catalysis. For this, we conjugated the R-groups of single amino acids to polyethylene glycol (PEG) chains. Through this work we have determined that the identity of the amino acid R-group is important in both the synthesis and the catalytic activity of the particles. For Pt nanoparticle synthesis, we found that the hydrophobicity of the functional groups affected their ability to interact well with the particles during nucleation and growth, and thus only the hydrophilic functional groups were capable of mediating the synthesis to produce well-defined faceted particles. With respect to ORR, we found distinct trends that showed that the inclusion of certain amino acids could significantly enhance catalysis---even at high polymer loadings. This work presents evidence that counters the common conception that organic capping ligands decrease catalytic activity; in fact activity may actually be

  4. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  5. Ruthenium(II) carbonyl complexes bearing CCC-pincer bis-(carbene) ligands: synthesis, structures and activities toward recycle transfer hydrogenation reactions.

    PubMed

    Naziruddin, Abbas Raja; Huang, Zhao-Jiunn; Lai, Wei-Chih; Lin, Wan-Jung; Hwang, Wen-Shu

    2013-09-28

    A new series of ruthenium(II) carbonyl complexes with benzene-based CCC-pincer bis-(carbene) ligands, [((R)CCC(R))Ru(CO)2(X)](0/+) and [((R)CCC(R))Ru(CO)(NN)](+) ((R)CCC(R) = 2,6-bis-(1-alkylimidazolylidene)benzene, R = Me or (n)Bu; X = I, Br, CH3CN, or 6-(aminomethyl)pyridine (ampy); NN = 2·CH3CN, or chelating ampy or bipyridine), was synthesized and fully characterized. X-Ray structure determinations revealed that these eight complexes have pseudo-octahedral configurations around the ruthenium center with the pincer ligand occupying three meridional sites. These complexes prove to be efficient precatalysts demonstrating very good activity and reusability for the transfer hydrogenation of ketones. PMID:23880878

  6. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  7. Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

    PubMed Central

    Wu, Xue; Xiu, Zhilong; Li, Guohui; Luo, Bing-Hao

    2013-01-01

    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 Ala252 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 Ala252 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. PMID:24116162

  8. A Dynamic Zn Site in Helicobacter pylori HypA: A Potential Mechanism for Metal-Specific Protein Activity

    SciTech Connect

    Kennedy,D.; Herbst, R.; Iwig, J.; Chivers, P.; Maroney, M.

    2007-01-01

    HypA is an accessory protein and putative metallochaperone that is critical for supplying nickel to the active site of NiFe hydrogenases. In addition to binding Ni(II), HypA is known to contain a Zn site that has been suggested to play a structural role. X-ray absorption spectroscopy has been used to show that the Zn site changes structure upon binding nickel, from a S{sub 3}(O/N)-donor ligand environment to an S{sub 4}-donor ligand environment. This provides a potential mechanism for discriminating Ni(II) from other divalent metal ions. The Ni(II) site is shown to be a six-coordinate complex composed of O/N-donors including two histidines. As such, it resembles the nickel site in UreE, a nickel metallochaperone involved in nickel incorporation into urease.

  9. Pilot study of PK 11195, a selective ligand for the peripheral-type benzodiazepine binding sites, in inpatients with anxious or depressive symptomatology.

    PubMed

    Ansseau, M; von Frenckell, R; Cerfontaine, J L; Papart, P

    1991-01-01

    PK 11195 is a selective ligand for the peripheral-type benzodiazepine binding sites which exhibits anti-conflict activity in animals. In a pilot open study, PK 11195 was administered to 10 psychiatric inpatients characterized by a rating of at least "moderate" for the item "felt loss of vitality" and a rating of at least "moderate" for the items "anxiety" and/or "inhibition of drive" from the psychopathological scale of the system developed by the Association for Methodology and Documentation in Psychiatry (AMDP). The duration of the study was two weeks, with an initial daily dose of 200 mg of PK 11195 which could be increased up to 400 mg. Patients were assessed weekly using the psychopathological and somatic AMDP scales and at days 0, 4, 7, and 14 using the Hamilton anxiety scale and a checklist of symptoms and side-effects. The results showed significant improvement in the AMDP factor scores related to somatic complaints, depression, anxiety, apathy-retardation, and psycho-organic symptoms. However, anxiolytic activity, confirmed on the Hamilton anxiety scale, remained moderate and reached maximum effect after one week. No side-effects, drowsiness in particular, were reported. This study therefore suggests a potential beneficial activity of PK 11195 on anxiety and inhibition, which merits further investigation in controlled studies. PMID:1849286

  10. Pincer Ligand Modifications To Tune the Activation Barrier for H2 Elimination in Water Splitting Milstein Catalyst.

    PubMed

    Sandhya, Karakkadparambil S; Remya, Geetha S; Suresh, Cherumuttathu H

    2015-12-01

    Modifications on the ligand environment of Milstein ruthenium(II) pincer hydride catalysts have been proposed to fine-tune the activation free energy, ΔG(⧧) for the key step of H2 elimination in the water splitting reaction. This study conducted at the B3LYP level of density functional theory including the solvation effect reveals that changing the bulky t-butyl group at the P-arm of the pincer ligand by methyl or ethyl group can reduce the ΔG(⧧) by a substantial margin, ∼ 10 kcal/mol. The reduction in the steric effect of the pincer ligand causes exothermic association of the water molecule to the metal center and leads to significant stabilization of all the subsequent reaction intermediates and the transition state compared to those of the original Milstein catalyst that promotes endothermic association of the water molecule. Though electron donating groups on the pyridyl unit of the pincer ligand are advantageous for reducing the activation barrier in the gas phase, the effect is only 1-1.4 kcal/mol compared to that of an electron withdrawing group. The absolute minimum of the electrostatic potential at the hydride ligand and carbonyl stretching frequency of the catalyst are useful parameters to gauge the effect of ligand environment on the H2 elimination step of the water splitting reaction. PMID:26575086

  11. Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin*

    PubMed Central

    Matsuyama, Take; Yamashita, Takahiro; Imai, Hiroo; Shichida, Yoshinori

    2010-01-01

    Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation. PMID:20042594

  12. Monoclonal Antibodies Specific for Human IgM Fc Receptor Inhibit Ligand-binding Activity

    PubMed Central

    Kubagawa, Yoshiki; Honjo, Kazuhito; Kang, Dong-Won

    2014-01-01

    A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcμR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcμR+ cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcμR+ cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcμR binding. Unlike control Jurkat cells, FcμR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcμR, thereby promoting apoptosis of FcμR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcμR. PMID:25545208

  13. N2 activation by an iron complex with a strong electron-donating iminophosphorane ligand.

    PubMed

    Suzuki, Tatsuya; Wasada-Tsutsui, Yuko; Ogawa, Takahiko; Inomata, Tomohiko; Ozawa, Tomohiro; Sakai, Yoichi; Fryzuk, Michael D; Masuda, Hideki

    2015-10-01

    A new tridentate cyclopentane-bridged iminophosphorane ligand, N-(2-diisopropylphosphinophenyl)-P,P-diisopropyl-P-(2-(2,6-diisopropylphenylamido)cyclopent-1-enyl)phosphoranimine (NpNPiPr), was synthesized and used in the preparation of a diiron dinitrogen complex. The reaction of the iron complex FeBr(NpNPiPr) with KC8 under dinitrogen yielded the dinuclear dinitrogen Fe complex [Fe(NpNPiPr)]2(μ-N2), which was characterized by X-ray analysis and resonance Raman and NMR spectroscopies. The X-ray analysis revealed a diiron complex bridged by the dinitrogen molecule, with each metal center coordinated by an NpNPiPr ligand and dinitrogen in a trigonal-monopyramidal geometry. The N–N bond length is 1.184(6) Å, and resonance Raman spectra indicate that the N–N stretching mode ν(14N2/15N2) is 1755/1700 cm–1. The magnetic moment of [Fe(NpNPiPr)]2(μ-N2) in benzene-d6 solution, as measured by 1H NMR spectroscopy by the Evans method, is 6.91μB (S = 3). The Mössbauer spectrum at 78 K showed δ = 0.73 mm/s and ΔEQ = 1.83 mm/s. These findings suggest that the iron ions are divalent with a high-spin configuration and that the N2 molecule has (N═N)2– character. Density functional theory calculations performed on [Fe(NpNPiPr)]2(μ-N2) also suggested that the iron is in a high-spin divalent state and that the coordinated dinitrogen molecule is effectively activated by π back-donation from the two iron ions (dπ) to the dinitrogen molecule (πx* and πy*). This is supported by cooperation between a large negative charge on the iminophosphorane ligand and strong electron donation and effective orbital overlap between the iron dπ orbitals and N2 π* orbitals supplied by the phosphine ligand. PMID:26135343

  14. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  15. Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP

    PubMed Central

    2015-01-01

    Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor. PMID:25648658

  16. Hydroxyeicosapentaenoic acids from the Pacific krill show high ligand activities for PPARs[S

    PubMed Central

    Yamada, Hidetoshi; Oshiro, Eriko; Kikuchi, Sayaka; Hakozaki, Mayuka; Takahashi, Hideyuki; Kimura, Ken-ichi

    2014-01-01

    PPARs regulate the expression of genes for energy metabolism in a ligand-dependent manner. PPARs can influence fatty acid oxidation, the level of circulating triglycerides, glucose uptake and insulin sensitivity. Here, we demonstrate that 5-hydroxyeicosapentaenoic acid (HEPE), 8-HEPE, 9-HEPE, 12-HEPE and 18-HEPE (hydroxylation products of EPA) obtained from methanol extracts of Pacific krill (Euphausia pacifica) can act as PPAR ligands. Two of these products, 8-HEPE and 9-HEPE, enhanced the transcription levels of GAL4-PPARs to a significantly greater extent than 5-HEPE, 12-HEPE, 18-HEPE, EPA, and EPA ethyl-ester. 8-HEPE also activated significantly higher transcription of GAL4-PPARα, GAL4-PPARγ, and GAL4-PPARδ than EPA at concentrations greater than 4, 64, and 64 μM, respectively. We also demonstrated that 8-HEPE increased the expression levels of genes regulated by PPARs in FaO, 3T3-F442A, and C2C12 cells. Furthermore, 8-HEPE enhanced adipogenesis and glucose uptake. By contrast, at the same concentrations, EPA showed weak or little effect, indicating that 8-HEPE was the more potent inducer of physiological effects. PMID:24668940

  17. Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity.

    PubMed

    Daburon, Sophie; Devaud, Christel; Costet, Pierre; Morello, Aurore; Garrigue-Antar, Laure; Maillasson, Mike; Hargous, Nathalie; Lapaillerie, Delphine; Bonneu, Marc; Dechanet-Merville, Julie; Legembre, Patrick; Capone, Myriam; Moreau, Jean-François; Taupin, Jean-Luc

    2013-01-01

    Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas. PMID:23326557

  18. Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site.

    PubMed

    Klotz, K N; Lohse, M J; Schwabe, U

    1988-11-25

    Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors. PMID:3182861

  19. Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

    PubMed

    Klein, Johanna C; Wild, Clarissa A; Lang, Stephan; Brandau, Sven

    2016-06-01

    Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy. PMID:27034235

  20. Ruthenium-based olefin metathesis catalysts bearing pH-responsive ligands: External control of catalyst solubility and activity

    NASA Astrophysics Data System (ADS)

    Balof, Shawna Lynn

    2011-12-01

    Sixteen novel, Ru-based olefin metathesis catalysts bearing pH responsive ligands were synthesized. The pH-responsive groups employed with these catalysts included dimethylamino (NMe2) modified NHC ligands as well as N-donor dimethylaminopyridine (DMAP) and 3-(o-pyridyl)propylidene ligands. These pH-responsive ligands provided the means by which the solubility and/or activity profiles of the catalysts produced could be controlled via acid addition. The main goal of this dissertation was to design catalyst systems capable of performing ring opening metathesis (ROMP) and ring closing metathesis (RCM) reactions in both organic and aqueous media. In an effort to quickly gain access to new catalyst structures, a template synthesis for functionalized NHC ligand precursors was designed, in addition to other strategies, to obtain ligand precursors with ancillary NMe2 groups. Kinetic studies for the catalysts produced from these precursors showed external control of catalyst solubility was afforded via protonation of the NMe2 groups of their NHC ligands. Additionally, this protonation afforded external control of catalyst propagation rates for several catalysts. This is the first known independent external control for the propagation rates of ROMP catalysts. The incorporation of pH-responsive N-donor ligands into catalyst structures also provided the means for the external control of metathesis activity, as the protonation of these ligands resulted in an increased initiation rate based on their fast and irreversible dissociation from the metal center. The enhanced external control makes these catalysts applicable to a wide range of applications, some of which have been explored by us and/or through collaboration. Three of the catalysts designed showed remarkable metathesis activity in aqueous media. These catalysts displayed comparable RCM activity in aqueous media to a class of water-soluble catalysts reported by Grubbs et al., considered to be the most active catalyst for

  1. Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity.

    PubMed

    Huan, Jianya Y; Meza-Romero, Roberto; Mooney, Jeffery L; Chou, Yuan K; Edwards, David M; Rich, Cathleen; Link, Jason M; Vandenbark, Arthur A; Bourdette, Dennis N; Bächinger, Hans-Peter; Burrows, Gregory G

    2005-01-01

    Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. PMID:22973070

  2. Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

    PubMed Central

    Huan, Jianya Y; Meza-Romero, Roberto; Mooney, Jeffery L; Chou, Yuan K; Edwards, David M; Rich, Cathleen; Link, Jason M; Vandenbark, Arthur A; Bourdette, Dennis N; Bächinger, Hans-Peter; Burrows, Gregory G

    2012-01-01

    Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. PMID:22973070

  3. The anticonvulsant actions of σ receptor ligands in the Mg2+-free model of epileptiform activity in rat hippocampal slices

    PubMed Central

    Thurgur, Claire; Church, John

    1998-01-01

    those of the established anticonvulsant adenosine; in contrast, adenosine evoked an increase in PPF ratios.Because anticonvulsant activity was observed only at micromolar concentrations of the σ ligands tested, the results indicate that their anticonvulsant actions should not be ascribed to their occupancy, observed at nanomolar concentrations, of high-affinity σ binding sites. Rather, anticonvulsant activity more likely reflects functional NMDA receptor antagonism and/or blockade of high voltage-activated Ca2+ channels, effects which are associated with micromolar concentrations of the test compounds. Modulation of GABAergic inhibitory mechanisms may also contribute to the anticonvulsant properties of caramiphen. PMID:9692777

  4. C-H functionalization: thoroughly tuning ligands at a metal ion, a chemist can greatly enhance catalyst's activity and selectivity.

    PubMed

    Shul'pin, Georgiy B

    2013-09-28

    This brief essay consists of a few "exciting stories" devoted to relations within a metal-complex catalyst between a metal ion and a coordinated ligand. When, as in the case of a human couple, the rapport of the partners is cordial and a love cements these relations, a chemist finds an ideal married couple, in other words he obtains a catalyst of choice which allows him to functionalize C-H bonds very efficiently and selectively. Examples of such lucky marriages in the catalytic world of ions and ligands are discussed here. Activity of the catalyst is characterized by turnover number (TON) or turnover frequency (TOF) as well as by yield of a target product. Introducing a chelating N,N- or N,O-ligand to the catalyst molecule (this can be an iron or manganese derivative) sharply enhances its activity. However, the activity of vanadium derivatives (with additionally added to the solution pyrazinecarboxylic acid, PCA) as well as of various osmium complexes does not dramatically depend on the nature of ligands surrounding metal ions. Complexes of these metals are very efficient catalysts in oxidations with H2O2. Osmium derivatives are record-holders exhibiting extremely high TONs whereas vanadium complexes are on the second position. Finally, elegant examples of alkane functionalization on the ions of non-transition metals (aluminium, gallium etc.) are described when one ligand within the metal complex (namely, hydroperoxyl ligand HOO(-)) helps other ligand of this complex (H2O2 molecule coordinated to the metal) to disintegrate into two species, generating very reactive hydroxyl radical. Hydrogen peroxide molecule, even ligated to the metal ion, is perfectly stable without the assistance of the neighboring HOO(-) ligand. This ligand can be easily oxidized donating an electron to its partner ligand (H2O2). In an analogous case, when the central ion in the catalyst is a transition metal, this ion changing its oxidation state can donate an electron to the coordinated H2O2

  5. Molecular mechanisms of human IRE1 activation through dimerization and ligand binding

    PubMed Central

    Joshi, Amar; Newbatt, Yvette; McAndrew, P. Craig; Stubbs, Mark; Burke, Rosemary; Richards, Mark W.; Bhatia, Chitra; Caldwell, John J.; McHardy, Tatiana; Collins, Ian; Bayliss, Richard

    2015-01-01

    IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors. PMID:25968568

  6. Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density

    PubMed Central

    Fasoli, Ezio; Reyes, Yiaslin Ruiz; Guzman, Osiris Martinez; Rosado, Alexandra; Cruz, Vivian Rodriguez; Borges, Amaris; Martinez, Edmarie; Bansal, Vibha

    2013-01-01

    Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; ii) the affinity ligand (pABA) density on membrane surface (5–25 nmoles per cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi- component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14- atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmole of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in monocomponent system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0 nmoles per cm2) in mono- and multi- component systems for 7- as well as 14- atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use. PMID:23703544

  7. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand.

    PubMed

    Saini, P; Ganugula, R; Arora, M; Kumar, M N V Ravi

    2016-01-01

    The current methods for targeted drug delivery utilize ligands that must out-compete endogenous ligands in order to bind to the active site facilitating the transport. To address this limitation, we present a non-competitive active transport strategy to overcome intestinal barriers in the form of tunable nanosystems (NS) for transferrin receptor (TfR) utilizing gambogic acid (GA), a xanthanoid, as its ligand. The NS made using GA conjugated poly(lactide-co-glycolide) (PLGA) have shown non-competitive affinity to TfR evaluated in cell/cell-free systems. The fluorescent PLGA-GA NS exhibited significant intestinal transport and altered distribution profile compared to PLGA NS in vivo. The PLGA-GA NS loaded with cyclosporine A (CsA), a model peptide, upon peroral dosing to rodents led to maximum plasma concentration of CsA at 6 h as opposed to 24 h with PLGA-NS with at least 2-fold higher levels in brain at 72 h. The proposed approach offers new prospects for peroral drug delivery and beyond. PMID:27388994

  8. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand

    PubMed Central

    Saini, P.; Ganugula, R.; Arora, M.; Kumar, M. N. V. Ravi

    2016-01-01

    The current methods for targeted drug delivery utilize ligands that must out-compete endogenous ligands in order to bind to the active site facilitating the transport. To address this limitation, we present a non-competitive active transport strategy to overcome intestinal barriers in the form of tunable nanosystems (NS) for transferrin receptor (TfR) utilizing gambogic acid (GA), a xanthanoid, as its ligand. The NS made using GA conjugated poly(lactide-co-glycolide) (PLGA) have shown non-competitive affinity to TfR evaluated in cell/cell-free systems. The fluorescent PLGA-GA NS exhibited significant intestinal transport and altered distribution profile compared to PLGA NS in vivo. The PLGA-GA NS loaded with cyclosporine A (CsA), a model peptide, upon peroral dosing to rodents led to maximum plasma concentration of CsA at 6 h as opposed to 24 h with PLGA-NS with at least 2-fold higher levels in brain at 72 h. The proposed approach offers new prospects for peroral drug delivery and beyond. PMID:27388994

  9. Norfloxacin and N-Donor Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin Interaction, and Anti-Trypanosoma cruzi Activity

    PubMed Central

    Martins, Darliane A.; Gouvea, Ligiane R.; Muniz, Gabriel S. Vignoli; Louro, Sonia R. W.; Batista, Denise da Gama Jaen; Soeiro, Maria de Nazaré C.; Teixeira, Letícia R.

    2016-01-01

    Copper(II) complexes with the first-generation quinolone antibacterial agent norfloxacin containing a nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy) or 1,10-phenanthroline (phen) were prepared and characterized by IR, EPR spectra, molar conductivity, and elemental analyses. The experimental data suggest that norfloxacin was coordinated to copper(II) through the carboxylato and ketone oxygen atoms. The interaction of the copper(II) complexes with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated using fluorescence quenching of the tryptophan residues and copper(II) EPR spectroscopy. The results of fluorescence titration revealed that copper(II) complexes have a moderate ability to quench the intrinsic fluorescence of the albumins through a static quenching mechanism. EPR experiments showed that BSA and HSA Cu(II) sites compete with NOR for Cu(II)-bipy and Cu(II)-phen to form protein mixed-ligand complexes. Copper(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The tests performed using bloodstream trypomastigotes showed that the Cu(II)-N-donor precursors and the metal complexes were more active than the free fluoroquinolone. PMID:26924953

  10. Another Piece in the Fibrotic Puzzle: TSLP as a Novel Ligand for Fibrocyte Activation.

    PubMed

    Christmann, Romy Beatriz

    2016-02-01

    Thymic stromal lymphopoietin (TSLP) has emerged as an important cytokine in the pathogenesis of nonallergic diseases, especially in diseases that include fibrosis. It has been shown to be upregulated in both cutaneous and lung fibrotic conditions. Shin et al. report that TSLP may also play a role in the pathogenesis of keloids. The main mechanism of TSLP profibrotic effects is not as yet fully understood, although the data suggest that it involves collagen production through transforming growth factor-β, at least in the case of dermal fibroblasts. The authors also report that TSLP is able to activate fibrocytes, probably by inducing stromal cell-derived factor-1 (also termed CXCL12), one of its main ligands. These findings support the concept that TSLP plays a role in the development of fibrosis, and they should lead to mechanistic studies on TSLP profibrotic signaling. PMID:26802232

  11. Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    PubMed Central

    Luo, Lichao; Wang, Huashan; Fan, Chao; Liu, Sen

    2015-01-01

    Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. Here, we show that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells is regulated by multiple Wnt ligands that are produced by the niche. Our data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary. PMID:26008746

  12. Characterization and biological activities of two copper(II) complexes with dipropylenetriamine and diamine as ligands

    NASA Astrophysics Data System (ADS)

    AL-Noaimi, Mousa; Choudhary, Mohammad I.; Awwadi, Firas F.; Talib, Wamidh H.; Hadda, Taibi Ben; Yousuf, Sammer; Sawafta, Ashraf; Warad, Ismail

    2014-06-01

    Two new mixed-ligand copper(II) complexes, [Cu(dipn)(Nsbnd N)]Br2(1-2) [dipn = dipropylenetriamine, Nsbnd N = ethylenediamine (en) (1) and propylenediamine (pn) (2)], have been synthesized. These complexes were characterized by spectroscopic and thermal techniques. Crystal structure for 2 shows a distorted trigonal-bipyramidal geometry around Cu(II) ion with one solvate water molecule. Antimicrobial and antiproliferative assays were conducted to evaluate the biological activities of these complexes. The complexes exhibit a promising antimicrobial effect against an array of microbes at 200 μg/mL concentration. The antiproliferative assay shows a high potential of these complexes to target Human keratinocyte cell line with IC50 values of 155 and 152 μM. The absorption spectrum of 2 in water was modeled by time-dependent density functional theory (TD-DFT).

  13. Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

    PubMed

    Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

    2013-01-01

    One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism. PMID:24025780

  14. Antimalarial activity of ruthenium(II) and osmium(II) arene complexes with mono- and bidentate chloroquine analogue ligands.

    PubMed

    Ekengard, Erik; Glans, Lotta; Cassells, Irwin; Fogeron, Thibault; Govender, Preshendren; Stringer, Tameryn; Chellan, Prinessa; Lisensky, George C; Hersh, William H; Doverbratt, Isa; Lidin, Sven; de Kock, Carmen; Smith, Peter J; Smith, Gregory S; Nordlander, Ebbe

    2015-11-28

    Eight new ruthenium and five new osmium p-cymene half-sandwich complexes have been synthesized, characterized and evaluated for antimalarial activity. All complexes contain ligands that are based on a 4-chloroquinoline framework related to the antimalarial drug chloroquine. Ligands HL(1-8) are salicylaldimine derivatives, where HL(1) = N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine, and HL(2-8) contain non-hydrogen substituents in the 3-position of the salicylaldimine ring, viz. F, Cl, Br, I, NO2, OMe and (t)Bu for HL(2-8), respectively. Ligand HL(9) is also a salicylaldimine-containing ligand with substitutions in both 3- and 5-positions of the salicylaldimine moiety, i.e. N-(2-((2-hydroxy-3,5-di-tert-butylphenyl)methyl-imino)ethyl)-7-chloroquinolin-4-amine, while HL(10) is N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine) The half sandwich metal complexes that have been investigated are [Ru(η(6)-cym)(L(1-8))Cl] (Ru-1-Ru-8, cym = p-cymene), [Os(η(6)-cym)(L(1-3,5,7))Cl] (Os-1-Os-3, Os-5, and Os-7), [M(η(6)-cym)(HL(9))Cl2] (M = Ru, Ru-HL(9); M = Os, Os-HL(9)) and [M(η(6)-cym)(L(10))Cl]Cl (M = Ru, Ru-10; M = Os, Os-10). In complexes Ru-1-Ru-8 and Ru-10, Os-1-Os-3, Os-5 and Os-7 and Os-10, the ligands were found to coordinate as bidentate N,O- and N,N-chelates, while in complexes Ru-HL(9) and Os-HL(9), monodentate coordination of the ligands through the quinoline nitrogen was established. The antimalarial activity of the new ligands and complexes was evaluated against chloroquine sensitive (NF54 and D10) and chloroquine resistant (Dd2) Plasmodium falciparum malaria parasite strains. Coordination of ruthenium and osmium arene moieties to the ligands resulted in lower antiplasmodial activities relative to the free ligands, but the resistance index is better for the ruthenium complexes compared to chloroquine. Overall, osmium complexes appeared to be less active than the corresponding ruthenium complexes. PMID:26491831

  15. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

  16. Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding.

    PubMed

    Blancato, Víctor S; Pagliai, Fernando A; Magni, Christian; Gonzalez, Claudio F; Lorca, Graciela L

    2016-01-01

    The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (K D = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni(2+), and Zn(2+) to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation. PMID:26903980

  17. Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding

    PubMed Central

    Blancato, Víctor S.; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio F.; Lorca, Graciela L.

    2016-01-01

    The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation. PMID:26903980

  18. Palladium(II) and platinum(II) derivatives of benzothiazoline ligands: Synthesis, characterization, antimicrobial and antispermatogenic activity

    NASA Astrophysics Data System (ADS)

    Sharma, Krishna; Singh, R. V.; Fahmi, Nighat

    2011-01-01

    A series of Pd(II) and Pt(II) complexes with two N ∩S donor ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and 6-nitro-3-(indolin-2-one)benzothiazoline, have been synthesized by the reaction of metal chlorides (PdCl 2 and PtCl 2) with ligands in 1:2 molar ratios. All the synthesized compounds were characterized by elemental analyses, melting point determinations and a combination of electronic, IR, 1H NMR and 13C NMR spectroscopic techniques for structure elucidation. In order to evaluate the effect of metal ions upon chelation, both the ligands and their complexes have been screened for their antimicrobial activity against the various pathogenic bacterial and fungal strains. The metal complexes have shown to be more antimicrobial against the microbial species as compared to free ligands. One of the ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and its corresponding palladium and platinum complexes have been tested for their antifertility activity in male albino rats. The marked reduction in sperm motility and density resulted in infertility by 62-90%. Significant alterations were found in biochemical parameters of reproductive organs in treated animals as compared to control group. It is concluded that all these effects may finally impair the fertility of male rats.

  19. [Integrin-ligands binding reaction upregulates the antioxidant activity of rabbit bronchial epithelial cells].

    PubMed

    Qin, X Q; Xiang, Y; Guan, C X; Zhang, C Q; Sun, X H

    2001-02-01

    Antioxidant activity of bronchial epithelial cells (BECs) plays an essential role in preventing the airway epithelium integrity from damage in structure and function. Integrin expressed by BECs is the receptor of extracellular matrix such as fibronectin (Fn), and it is involved in modulation of proliferation, differentiation and metabolism of the cells. In order to test the hypothesis that integrin-ligand binding reaction supports the ability of cells to withstand oxidant attack, the present study evaluated the antioxidant activity of primary cultured rabbit BECs treated with fibronectin or its sequence Arg-Gly-Asp (RGD peptide), by determining changes in the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) and in the level of glutathione (GSH). The results are as follows: (1) Fn (10 micrograms/ml) increased significantly the activity unit of GSH-Px (P < 0.05, n = 5), which was inhibited by calmodulin-inhibitor W7 (10(-5) mol/L) (P < 0.05). Both Fn (5-20 micrograms/ml) and RGD (15-60 micrograms/ml) showed a dose-dependent upregulatory effect (respectively r = 0.93 and r = 0.73). (2) Treatment with Fn increased SOD activity (P < 0.01, n = 7), which was abolished by W7 (P < 0.01). (3) Catalase activity was also stimulated by Fn (P < 0.05, n = 6) and reversed by W7 (P < 0.01). (4) A dose-dependent increase of GSH level was observed in both Fn (r = 0.82) and RGD treatment (r = 0.84). The data suggest that the binding of integrin with extracellular matrix can upregulate activity of antioxidant enzymes, and increase the content of GSH and improve the ability of BECs to resist oxidant injury. PMID:11354796

  20. Metal-ligand cooperation by aromatization-dearomatization: a new paradigm in bond activation and "green" catalysis.

    PubMed

    Gunanathan, Chidambaram; Milstein, David

    2011-08-16

    In view of global concerns regarding the environment and sustainable energy resources, there is a strong need for the discovery of new, green catalytic reactions. For this purpose, fresh approaches to catalytic design are desirable. In recent years, complexes based on "cooperating" ligands have exhibited remarkable catalytic activity. These ligands cooperate with the metal center by undergoing reversible structural changes in the processes of substrate activation and product formation. We have discovered a new mode of metal-ligand cooperation, involving aromatization-dearomatization of ligands. Pincer-type ligands based on pyridine or acridine exhibit such cooperation, leading to unusual bond activation processes and to novel, environmentally benign catalysis. Bond activation takes place with no formal change in the metal oxidation state, and so far the activation of H-H, C-H (sp(2) and sp(3)), O-H, and N-H bonds has been demonstrated. Using this approach, we have demonstrated a unique water splitting process, which involves consecutive thermal liberation of H(2) and light-induced liberation of O(2), using no sacrificial reagents, promoted by a pyridine-based pincer ruthenium complex. An acridine pincer complex displays unique "long-range" metal-ligand cooperation in the activation of H(2) and in reaction with ammonia. In this Account, we begin by providing an overview of the metal-ligand cooperation based on aromatization-dearomatization processes. We then describe a range of novel catalytic reactions that we developed guided by these new modes of metal-ligand cooperation. These reactions include the following: (1) acceptorless dehydrogenation of secondary alcohols to ketones, (2) acceptorless dehydrogenative coupling of alcohols to esters, (3) acylation of secondary alcohols by esters with dihydrogen liberation, (4) direct coupling of alcohols and amines to form amides and polyamides with liberation of dihydrogen, (5) coupling of esters and amines to form amides

  1. Synthesis, X-ray crystal structures and catecholase activity investigation of new chalcone ligands

    NASA Astrophysics Data System (ADS)

    Thabti, Salima; Djedouani, Amel; Rahmouni, Samra; Touzani, Rachid; Bendaas, Abderrahmen; Mousser, Hénia; Mousser, Abdelhamid

    2015-12-01

    The reaction of dehydroacetic acid DHA carboxaldehyde and RCHO derivatives (R = quinoleine-8-; indole-3-; pyrrol-2- and 4-(dimethylamino)phenyl - afforded four new chalcone ligands (4-hydroxy-6-methyl-3-[(2E)-3-quinolin-8-ylprop-2-enoyl]-2H-pyran-2-one) L1, (4-hydroxy-3-[(2E)-3-(1H-indol-3-yl)prop-2-enoyl]-6-methyl-2H-pyran-2-one) L2, (4-hydroxy-6-methyl-3-[(2E)-3-(1H-pyrrol-2-yl)prop-2-enoyl]-2H-pyran-2-one) L3, and (3-{(2E)-3-[4-(dimethylamino)phenyl]prop-2-enoyl}-4-hydroxy-6-methyl-2H-pyran-2-one) L4. L3 and L4 were characterized by X-ray crystallography. Molecules crystallize with four and two molecules in the asymmetric unit, respectively and adopt an E conformation about the Cdbnd C bond. Both structures are stabilized by an extended network O-H … O. Furthermore, N-H … O and C-H … O hydrogen bonds are observed in L3 and L4 structures, respectively. The in situ generated copper (II) complexes of the four compounds L1, L2, L3 and L4 were examined for their catalytic activities and were found to catalyze the oxidation reaction of catechol to o-quinone under atmospheric dioxygen. The rates of this oxidation depend on three parameters: ligand, ion salts and solvent nature and the combination L2[Cu (CH3COO)2] leads to the faster catalytic process.

  2. Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

    PubMed

    Vasta, James D; Raines, Ronald T

    2016-06-14

    Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits. PMID:27183028

  3. Porous capsules with a large number of active sites: nucleation/growth under confined conditions.

    PubMed

    Garai, Somenath; Rubčić, Mirta; Bögge, Hartmut; Gouzerh, Pierre; Müller, Achim

    2015-03-01

    This work deals with the generation of large numbers of active sites and with ensuing nucleation/ growth processes on the inside wall of the cavity of porous nanocapsules of the type (pentagon)12(linker)30≡{(Mo(VI))Mo(VI)5}12{Mo(V)2(ligand)}30. A first example refers to sulfur dioxide capture through displacement of acetate ligands, while the grafted sulfite ligands are able to trap {MoO3H}(+) units thereby forming unusual {(O2SO)3MoO3H}(5-) assemblies. A second example relates to the generation of open coordination sites through release of carbon dioxide upon mild acidification of a carbonate-type capsule. When the reaction is performed in the presence of heptamolybdate ions, MoO4(2-) ions enter the cavity where they bind to the inside wall while forming new types of polyoxomolybdate architectures, thereby extending the molybdenum oxide skeleton of the capsule. Parallels can be drawn with Mo-storage proteins and supported MoO3 catalysts, making the results relevant to molybdenum biochemistry and to catalysis. PMID:25653204

  4. A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

    PubMed Central

    Melia, T J; Cowan, C W; Angleson, J K; Wensel, T G

    1997-01-01

    Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation. PMID:9414230

  5. Synthesis, spectroscopic characterization and antimicrobial activity of mono-, bi- and tri-nuclear metal complexes of a new Schiff base ligand

    NASA Astrophysics Data System (ADS)

    Shebl, Magdy; Khalil, Saied M. E.; Ahmed, Saleh A.; Medien, Hesham A. A.

    2010-09-01

    Condensation of o-acetoacetylphenol and 1,2-diaminopropane in 1:1 molar ratio under condition of high dilution yielded the mono-condensed dibasic Schiff base ligand with a N 2O 2 donors. The mono-condensed ligand has been used for further condensation with 2-hydroxy-5-nitrobenzaldehyde to obtain the new asymmetrical dicompartmental Schiff base ligand, H 3L, with N 2O 3 donors. The structure of the ligand was elucidated by analytical and spectroscopic tools (IR, 1H and 13C NMR spectra) which indicated that the coordinating sites are oxygen atoms of the phenolic OH groups, nitrogen atoms of the azomethine groups and the oxygen atom of the ketonic group. Reactions of the ligand with metal salts yielded mono- and homo-bi-nuclear complexes formulated as [M(HL)], where M dbnd Co(II), Ni(II) and Cu(II), [Fe(H 2L)Cl 2(H 2O)]ṡ2½H 2O, [Fe 2(HL)(ox)Cl 3(H 2O) 2]ṡ5H 2O, [UO 2(H 2L)(OAc)(H 2O) 2], [VO(H 3L)(SO 4)(H 2O)]ṡH 2O, [M 2(L)Cl(H 2O) 2]ṡ½H 2O, where M dbnd Co(II) and Ni(II) and [Cu(H 2L)Cl]. The mononuclear Ni(II) complex, [Ni(HL)], was used to synthesize homo- and hetero-bi- and tri-nuclear complexes with the molecular formulae [Ni 2(L)Cl(H 2O) 2], [Ni 2(L) 2FeCl(H 2O)]ṡH 2O and [Ni 2(HL) 2CoCl 2]. The structures of the complexes were characterized by various techniques such as elemental and thermal analyses, IR, 1H and 13C NMR, mass and electronic spectra as well as conductivity and magnetic moment measurements. Square-planar and octahedral geometries are suggested for the Cu(II), Co(II) and Ni(II) complexes, octahedral geometry for the Fe(III) and VO 2+ complexes while uranium(VI) ion is octa-coordinated in its complex. The Schiff base and its metal complexes were evaluated for antimicrobial activity against Gram positive bacteria ( Staphylococcus aureus), Gram negative bacteria ( Escherichia coli) and fungi ( Candida albicans and Aspergillus flavus). The ligand and some of its complexes were found to be biologically active.

  6. Design of a Water Soluble Fluorescent 3-Hydroxy-4-Pyridinone Ligand Active at Physiological pH Values.

    PubMed

    Leite, Andreia; Silva, Ana M G; Coutinho, Catarina; Cunha-Silva, Luís; de Castro, Baltazar; Rangel, Maria

    2016-09-01

    In the present work we report the structure and the spectroscopic characterization of a new fluorescent 3-hydroxy-4-pyridinone ligand D-3,4-HPO. The synthesis of the compound was performed in two steps, which involve the reaction of the commercially available fluorophore dansyl chloride with a 3-hydroxy-4-pyridinone chelating unit and further deprotection. The new fluorescent chelator was characterized in the solid state by single-crystal X-ray diffraction and in solution by NMR, MS, absorption and fluorescence spectroscopies. The analysis of the variation of the absorption spectrum with pH allowed the determination of four pK a values (pK a1  = 3.50, pK a2  = 4.50, pK a3  = 9.60, pK a4  = 10.20) and establishment of the corresponding distribution diagram. The study of the fluorescence properties of the ligand show that in the pH range between 4 and 9 the fluorescence intensity is constant and has its maximum value thus allowing its further use at physiological pH values. The interaction of the ligand with copper(II) was accessed by fluorescence spectroscopy in MOPS buffer and the results show that the presence of copper(II) quenches the fluorescence of the ligand in ca 94 % at a ligand: metal ratio of 2:1. The latter result is consistent with the formation of a copper(II) complex with the bidentate ligand, as confirmed by the EPR spectroscopy. Graphical Abstract New water soluble fluorescent ligand active at physiological pH values. PMID:27357392

  7. Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

    PubMed

    Chavez-Dueñas, Lucia; Serapio-Palacios, Antonio; Nava-Acosta, Raul; Navarro-Garcia, Fernando

    2016-07-01

    Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed β-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent β-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed β-helical structure. Interestingly, Pet lacking the d2 domain (PetΔd2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (PetΔd1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, PetΔd1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, PetΔd2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin. PMID:27113356

  8. Different AhR binding sites of diterpenoid ligands from Andrographis paniculata caused differential CYP1A1 induction in primary culture in mouse hepatocytes.

    PubMed

    Chatuphonprasert, Waranya; Remsungnen, Tawun; Nemoto, Nobuo; Jarukamjorn, Kanokwan

    2011-12-01

    Andrographis paniculata has been employed as a folklore remedy. Andrographolide (Andro), 14-deoxy-11,12-didehydroandrographolide (DHA), andrographiside (AS), and neoandrographolide (Neo), are major diterpenoids isolated from this plant. In the present study, influence of the four diterpenoids on CYP1A1 mRNA expression was investigated in primary cultured mouse hepatocytes. Additionally, binding of these compounds to aryl hydrocarbon receptor (AhR) was examined using molecular docking analysis to clarify mechanism of CYP1A1 induction. Andro and DHA induced CYP1A1 expression by itself, and co-treatment with a CYP1A1 inducer (BNF, beta-naphthoflavone) showed a synergistic increase of CYP1A1 expression. Andro demonstrated higher enhancing activity than DHA at every similar concentration. On the other hand, Neo suppressed BNF-induced CYP1A1 expression, but AS did not modify the induction. Results from molecular docking analysis of BNF and four diterpenoids on ligand binding domain of AhR were consistent with levels of CYP1A1 mRNA expressions. Furthermore, difference of binding sites of BNF in the presence of diterpenoids might affect the synergism or inhibition of CYP1A1 expression. These results suggest that use of A. paniculata as a health supplement should be concerned in term of herb-drugs interactions or risk of carcinogenesis, according to its ability to influence CYP1A1 expression. PMID:21963808

  9. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  10. Activity landscape modeling of PPAR ligands with dual-activity difference maps.

    PubMed

    Méndez-Lucio, Oscar; Pérez-Villanueva, Jaime; Castillo, Rafael; Medina-Franco, José L

    2012-06-01

    Activation of peroxisome proliferator-activated receptor (PPAR) subtypes offers a promising strategy for the treatment of diabetes mellitus and metabolic diseases. Selective and dual PPAR agonists have been developed and the systematic characterization of their structure-activity relationships (SAR) is of major significance. Herein, we report a systematic description of the SAR of 168 compounds screened against the three PPAR subtypes using the principles of activity landscape modeling. As part of our effort to develop and apply chemoinformatic tools to navigate through activity landscapes, we employed consensus dual-activity difference maps recently reported. The analysis is based on pairwise relationships of potency difference and structure-similarity which were calculated from the combination of four different 2D and 3D structure representations. Dual-activity difference maps uncovered regions in the landscape with similar SAR for two or three receptor subtypes as well as regions with inverse SAR, that is, changes in structure that increase activity for one subtype but decrease activity for the other subtype. Analysis of pairs of compounds with high structure similarity revealed the presence of single-, dual-, and 'pan-receptor' activity cliffs, that is, small changes in structure with high changes in potency for one, two, or three receptor subtypes, respectively. Single-, dual-, and pan-receptor scaffold hops are also discussed. The analysis of the chemical structures of selected data points reported in this paper points to specific structural features that are helpful for the design of new PPAR agonists. The approach presented in this work is general and can be extended to analyze larger data sets. PMID:22564380

  11. Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors.

    PubMed

    Tränkle, Christian; Weyand, Oliver; Voigtländer, Uta; Mynett, Anita; Lazareno, Sebastian; Birdsall, Nigel J M; Mohr, Klaus

    2003-07-01

    An optimized assay for the binding of [3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the allosteric site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated. PMID:12815174

  12. A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery

    PubMed Central

    Xie, Lei; Xie, Li; Bourne, Philip E.

    2009-01-01

    Functional relationships between proteins that do not share global structure similarity can be established by detecting their ligand-binding-site similarity. For a large-scale comparison, it is critical to accurately and efficiently assess the statistical significance of this similarity. Here, we report an efficient statistical model that supports local sequence order independent ligand–binding-site similarity searching. Most existing statistical models only take into account the matching vertices between two sites that are defined by a fixed number of points. In reality, the boundary of the binding site is not known or is dependent on the bound ligand making these approaches limited. To address these shortcomings and to perform binding-site mapping on a genome-wide scale, we developed a sequence-order independent profile–profile alignment (SOIPPA) algorithm that is able to detect local similarity between unknown binding sites a priori. The SOIPPA scoring integrates geometric, evolutionary and physical information into a unified framework. However, this imposes a significant challenge in assessing the statistical significance of the similarity because the conventional probability model that is based on fixed-point matching cannot be applied. Here we find that scores for binding-site matching by SOIPPA follow an extreme value distribution (EVD). Benchmark studies show that the EVD model performs at least two-orders faster and is more accurate than the non-parametric statistical method in the previous SOIPPA version. Efficient statistical analysis makes it possible to apply SOIPPA to genome-based drug discovery. Consequently, we have applied the approach to the structural genome of Mycobacterium tuberculosis to construct a protein–ligand interaction network. The network reveals highly connected proteins, which represent suitable targets for promiscuous drugs. Contact: lxie@sdsc.edu PMID:19478004

  13. The extracellular loop 2 (ECL2) of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

    PubMed

    Wifling, David; Bernhardt, Günther; Dove, Stefan; Buschauer, Armin

    2015-01-01

    In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R. PMID:25629160

  14. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  15. Synthesis, characterization and antibacterial activity of mixed ligand dioxouranium complexes of 8-hydroxyquinoline and some amino acids.

    PubMed

    Patil, Sunil S; Shaikh, Manzoor M

    2012-01-01

    Mixed ligand complexes of dioxouranium(VI) of the type [UO2(Q)(L)-2H2O] have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and N- and/or O- donor amino acids (HL) such as L-lysine, L-aspartic acid and L-cysteine as secondary ligands. The metal complexes have been characterized on the basis of elemental analysis, electrical conductance, room temperature magnetic susceptibility measurements, spectral and thermal studies. The electrical conductance studies of the complexes in DMF in 10(-3) M concentration indicate their non-electrolytic nature. Room temperature magnetic susceptibility measurements revealed diamagnetic nature of the complexes. Electronic absorption spectra of the complexes show intra-ligand and charge transfer transitions, respectively. Bonding of the metal ion through N- and O- donor atoms of the ligands is revealed by IR studies and the chemical environment of the protons is also confirmed by NMR studies. The thermal analysis data of the complexes indicate the presence of coordinated water molecules. The agar cup and tube dilution methods have been used to study the antibacterial activity of the complexes against the pathogenic bacteria S. aureus, C. diphtherinae, S. typhi and E. coli. PMID:22876610

  16. Epidermal Growth Factor Receptor Dimerization and Activation Require Ligand-Induced Conformational Changes in the Dimer Interface

    PubMed Central

    Dawson, Jessica P.; Berger, Mitchell B.; Lin, Chun-Chi; Schlessinger, Joseph; Lemmon, Mark A.; Ferguson, Kathryn M.

    2005-01-01

    Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively “buttressing” the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization. PMID:16107719

  17. Peroxisome proliferator-activated receptor gamma ligands induce growth inhibition and apoptosis of human B lymphocytic leukemia.

    PubMed

    Zang, Chuanbing; Liu, Hongyu; Posch, Maximilian G; Waechter, Maries; Facklam, Margit; Fenner, Martin H; Ruthardt, Martin; Possinger, Kurt; Phillip Koeffler, H; Elstner, Elena

    2004-04-01

    This study examined the expression and structural intactness of peroxisome proliferator-activated receptor gamma (PPARgamma) in human acute lymphocytic leukemia (ALL) cells and determined the effect of PPARgamma ligands on growth and apoptosis of these cells. We noted that all lymphocytic leukemia cell lines expressed PPARgamma and no PPARgamma mutations were found in these cell lines as indicated by SSCP analysis. Effect of the PPARgamma ligands on the proliferation, differentiation and apoptosis of B type ALL cells was further examined. Treatment of these cells with the PPARgamma ligands Pioglitazone (PGZ) and 15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) resulted in growth inhibition in a dose-dependent manner which was associated with a G1 to S cell cycle arrest. However, this effect appeared to be PPARgamma-independent since several PPARgamma antagonists could not reverse this effect. No differentiation was induced by this treatment. Four out of five cell lines underwent apoptosis after culture with the PPARgamma ligands. This effect was partially caspase-dependent because a pan-caspase inhibitor partially reversed this effect. In conclusion, our results suggest that PPARgamma ligands may offer a new therapeutic approach to aid in the treatment of ALL. PMID:15109539

  18. Inferring Active and Prognostic Ligand-Receptor Pairs with Interactions in Survival Regression Models

    PubMed Central

    Ruggeri, Christina; Eng, Kevin H

    2014-01-01

    Modeling signal transduction in cancer cells has implications for targeting new therapies and inferring the mechanisms that improve or threaten a patient’s treatment response. For transcriptome-wide studies, it has been proposed that simple correlation between a ligand and receptor pair implies a relationship to the disease process. Statistically, a differential correlation (DC) analysis across groups stratified by prognosis can link the pair to clinical outcomes. While the prognostic effect and the apparent change in correlation are both biological consequences of activation of the signaling mechanism, a correlation-driven analysis does not clearly capture this assumption and makes inefficient use of continuous survival phenotypes. To augment the correlation hypothesis, we propose that a regression framework assuming a patient-specific, latent level of signaling activation exists and generates both prognosis and correlation. Data from these systems can be inferred via interaction terms in survival regression models allowing signal transduction models beyond one pair at a time and adjusting for other factors. We illustrate the use of this model on ovarian cancer data from the Cancer Genome Atlas (TCGA) and discuss how the finding may be used to develop markers to guide targeted molecular therapies. PMID:25657571

  19. Elevated autocrine chemokine ligand 18 expression promotes oral cancer cell growth and invasion via Akt activation

    PubMed Central

    Hong, Yun; Wu, Tong; Chen, Xiaobing; Xia, Juan; Cheng, Bin

    2016-01-01

    Chemokine (C-C motif) ligand 18 (CCL18) has been implicated in the pathogenesis and progression of various cancers; however, in oral squamous cell carcinoma (OSCC), the role of CCL18 is unknown. In this study, we found that CCL18 was overexpressed in primary OSCC tissues and was associated with an advanced clinical stage. CCL18 was found in both the cytoplasm and cell membrane of OSCC cells and was predominantly produced by cancer epithelial cells, as opposed to tumor-infiltrating macrophages. In vitro studies indicated that the effects of endogenous CCL18 on OSCC cell growth, migration, and invasion could be blocked by treatment with a neutralizing anti-CCL18 antibody or CCL18 knockdown, while exogenous recombinant CCL18 (rCCL18) rescued those effects. Akt was activated in rCCL18-treated OSCC cells, while LY294002, a pan-PI3K inhibitor, abolished both endogenous and exogenous CCL18-induced OSCC cell invasion. In vivo, LY294002 treatment attenuated rCCL18-induced OSCC cell growth. Our results indicate that CCL18 acts in an autocrine manner via Akt activation to stimulate OSCC cell growth and invasion during OSCC progression. They also provide a potential therapeutic target for the treatment of oral cancer. PMID:26919103

  20. Discovering new agents active against methicillin-resistant Staphylococcus aureus with ligand-based approaches.

    PubMed

    Wang, Ling; Le, Xiu; Li, Long; Ju, Yingchen; Lin, Zhongxiang; Gu, Qiong; Xu, Jun

    2014-11-24

    To discover new agents active against methicillin-resistant Staphylococcus aureus (MRSA), in silico models derived from 5451 cell-based anti-MRSA assay data were developed using four machine learning methods, including naïve Bayesian, support vector machine (SVM), recursive partitioning (RP), and k-nearest neighbors (kNN). A total of 876 models have been constructed based on physicochemical descriptors and fingerprints. The overall predictive accuracies of the best models exceeded 80% for both training and test sets. The best model was employed for the virtual screening of anti-MRSA compounds, which were then validated by a cell-based assay using the broth microdilution method with three types of highly resistant MRSA strains (ST239, ST5, and 252). A total of 12 new anti-MRSA agents were confirmed, which had MIC values ranging from 4 to 64 mg/L. This work proves the capacity of combined multiple ligand-based approaches for the discovery of new agents active against MRSA with cell-based assays. We think this work may inspire other lead identification processes when cell-based assay data are available. PMID:25375651

  1. Photo biological activation of NSO donor mixed-ligand complexes: in vivo and preclinical perspectives.

    PubMed

    Selvaganapathy, Muthusamy; Pravin, Narayanaperumal; Pothiraj, Krishnan; Raman, Natarajan

    2014-09-01

    Pyrazolone incorporating N-acetylcysteine (NAC) mixed-ligand complexes are described as promising anti-inflammatory, anticonvulsant, SODs mimetic and cytotoxic compounds possibly due to its antioxidant profile. In this study, we have evaluated the pharmacologic activity, antioxidant and toxicological profiles of compounds (1-6). Among them, compounds 1 and 4 were haemobiocompatible than the others. Both complexes 1 and 4 display efficient photo-nuclease activity upon irradiation with UV-A light of 365 nm and red light of 647 nm as compared with others. Mechanistic studies reveal that the DNA cleavage oxidative pathway involves H2O2 and singlet oxygen as the reactive oxygen species. Interestingly, both compounds 1 and 4, show non-toxic effects in vitro to human normal lymphocyte cells, revealing that they are selective in killing only the cancer cells as expected for a better drug. In addition, considering the safety profile, these compounds are promising as preventive and/or therapeutic agents against oxidative damage. PMID:24995992

  2. Ultrafast time-resolved IR studies of protein-ligand interactions.

    PubMed

    Lim, Manho; Anfinrud, Philip A

    2005-01-01

    Time-resolved mid-IR spectroscopy combines molecular sensitivity with ultrafast capability to incisively probe protein-ligand interactions in model heme proteins. Highly conserved residues near the heme binding site fashion a ligand-docking site that mediates the transport of ligands to and from the binding site. We employ polarization anisotropy measurements to probe the orientation and orientational distribution of CO when bound to and docked near the active binding site, as well as the dynamics of ligand trapping in the primary docking site. In addition, we use more conventional transient absorption methods to probe the dynamics of ligand escape from this site, as well as the ultrafast dynamics of NO geminate recombination with the active binding site. The systems investigated include myoglobin, hemoglobin, and microperoxidase. PMID:15940001

  3. Binding hotspots on K-Ras: consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis

    PubMed Central

    Prakash, Priyanka; Hancock, John F.; Gorfe, Alemayehu A.

    2015-01-01

    We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in non-pocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu. PMID:25740554

  4. Current Challenges for Modeling Enzyme Active Sites by Biomimetic Synthetic Diiron Complexes

    PubMed Central

    Friedle, Simone; Reisner, Erwin; Lippard, Stephen J.

    2010-01-01

    This tutorial review describes recent progress in modeling the active sites of carboxylate-rich non-heme diiron enzymes that activate dioxygen to carry out several key reactions in nature. The chemistry of soluble methane monooxygenase, which catalyzes the selective oxidation of methane to methanol, is of particular interest for (bio)technological applications. Novel synthetic diiron complexes that mimic structural, and, to a lesser extent, functional features of these diiron enzymes are discussed. The chemistry of the enzymes is also briefly summarized. A particular focus of this review is on models that mimic characteristics of the diiron systems that were previously not emphasized, including systems that contain (i) aqua ligands, (ii) different substrates tethered to the ligand framework, (iii) dendrimers attached to carboxylates to mimic the protein environment, (iv) two N-donors in a syn-orientation with respect to the iron-iron vector, and (v) a N-rich ligand environment capable of accessing oxygenated high-valent diiron intermediates. PMID:20485834

  5. New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: Spectral, magnetic, thermal and anticancer activity

    NASA Astrophysics Data System (ADS)

    El-Boraey, Hanaa A.; EL-Gammal, Ohyla A.

    2015-03-01

    Novel tetraamidemacrocyclic 15-membered ligand [L] i.e. naphthyl-dibenzo[1,5,9,12]tetraazacyclopentadecine-6,10,11,15-tetraoneand its transition metal complexes with Fe(II), Co(II), Ni(II), Cu(II), Ru(III) and Pd(II) have been synthesized and characterized by elemental analysis, spectral, thermal as well as magnetic and molar conductivity measurements. On the basis of analytical, spectral (IR, MS, UV-Vis, 1H NMR and EPR) and thermal studies distorted octahedral or square planar geometry has been proposed for the complexes. The antitumor activity of the synthesized ligand and some complexes against human breast cancer cell lines (MCF-7) and human hepatocarcinoma cell lines (HepG2) has been studied. The complexes (IC50 = 2.27-2.7, 8.33-31.1 μg/mL, respectively) showed potent antitumor activity, towards the former cell lines comparable with their ligand (IC50 = 13, 26 μg/mL, respectively). The results show that the activity of the ligand towards breast cancer cell line becomes more pronounced and significant when coordinated to the metal ion.

  6. Oxidorhenium(V) Complexes with Tetradentate Iminophenolate Ligands: Influence of Ligand Flexibility on the Coordination Motif and Oxygen-Atom-Transfer Activity.

    PubMed

    Zwettler, Niklas; Schachner, Jörg A; Belaj, Ferdinand; Mösch-Zanetti, Nadia C

    2016-06-20

    The synthesis of oxidorhenium(V) complexes 1-3 coordinated by tetradentate iminophenolate ligands H2L1-H2L3 bearing backbones of different rigidity (alkyl, cycloalkyl, and phenyl bridges) allows for the formation of distinct geometric isomers, including a symmetric trans-oxidochlorido coordination motif in complex 3. The complex employing a cycloalkyl-bridged ligand (2) of intermediate rigidity exhibits an interesting solvent- and temperature-dependent equilibrium between a symmetric (trans) isomer and an asymmetric (cis) isomer in solution. The occurrence of a symmetric isomer for 2 and 3 is confirmed by single-crystal X-ray diffraction analysis. Chlorido abstraction from 2 with AgOTf yields the corresponding cationic complex 2a, which does not exhibit an isomeric equilibrium in solution but adopts the isomeric form predominant for 2 in a given solvent. All complexes were, furthermore, employed in three benchmark oxygen-atom-transfer (OAT) reactions, namely, the reduction of perchlorate, the epoxidation of cyclooctene, and OAT from dimethyl sulfoxide (DMSO) to triphenylphosphane (PPh3), to assess the influence of the isomeric structure on the reactivity in these reactions. In perchlorate reduction, a clear structural influence was observed, where the trans arrangement in 3 led to the complete absence of activity. In the epoxidation reaction, all complexes led to comparable epoxide yields, albeit higher catalytic activity but lower overall stability of the catalysts with a trans arrangement was observed. In OAT from DMSO to PPh3, also a clear structural dependence was observed, where the trans complex 3 led to full phosphane conversion with an excess of oxidant, while the cis compound 1 was completely inactive. PMID:27251591

  7. Targeting Large Kinase Active Site with Rigid, Bulky Octahedral Ruthenium Complexes

    SciTech Connect

    Maksimoska, Jasna; Feng, Li; Harms, Klaus; Yi, Chunling; Kissil, Joseph; Marmorstein, Ronen; Meggers, Eric

    2009-09-02

    A strategy for targeting protein kinases with large ATP-binding sites by using bulky and rigid octahedral ruthenium complexes as structural scaffolds is presented. A highly potent and selective GSK3 and Pim1 half-sandwich complex NP309 was successfully converted