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Sample records for lipid binding site

  1. A novel lipid binding site formed by the MAP kinase insert in p38 alpha.

    PubMed

    Diskin, Ron; Engelberg, David; Livnah, Oded

    2008-01-01

    The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38 alpha MAP kinase in complex with n-octyl-beta-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38 alpha in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38 alpha catalytic activity towards a preferred phenotype. PMID:17999933

  2. Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor

    PubMed Central

    2016-01-01

    Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. −40 to −4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins. PMID:27109430

  3. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    PubMed

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

  4. Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

    PubMed Central

    Weber, Daniel K.; Yao, Shenggen; Rojko, Nejc; Anderluh, Gregor; Lybrand, Terry P.; Downton, Matthew T.; Wagner, John; Separovic, Frances

    2015-01-01

    Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-15N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation. PMID:25902438

  5. Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

    PubMed

    Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

    2016-07-01

    Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions. PMID:27324153

  6. Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

    PubMed Central

    Urbanek, Agnieszka N.; Allwood, Ellen G.; Smith, Adam P.; Booth, Wesley I.; Ayscough, Kathryn R.

    2015-01-01

    During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis. PMID:26312755

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

    PubMed Central

    Martin, Shaun; van Veen, Sarah; Holemans, Tine; Demirsoy, Seyma; van den Haute, Chris; Baekelandt, Veerle; Agostinis, Patrizia; Eggermont, Jan; Vangheluwe, Peter

    2016-01-01

    The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9) is implicated in Parkinson's disease (PD) and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA) and phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2), which modulate ATP13A2 activity under cellular stress conditions. Here, we analyzed stable human SHSY5Y cell lines overexpressing wild-type (WT) or ATP13A2 mutants in which three N-terminal lipid binding sites (LBS1–3) were mutated. We explored the regulatory role of LBS1–3 in the cellular protection by ATP13A2 against mitochondrial stress induced by rotenone and found that the LBS2-3 mutants displayed an abrogated protective effect. Moreover, in contrast to WT, the LBS2 and LBS3 mutants responded poorly to pharmacological inhibition of, respectively, PI(3,5)P2 and PA formation. We further demonstrate that PA and PI(3,5)P2 are also required for the ATP13A2-mediated protection against the toxic metals Mn2+, Zn2+, and Fe3+, suggesting a general lipid-dependent activation mechanism of ATP13A2 in various PD-related stress conditions. Our results indicate that the ATP13A2-mediated protection requires binding of PI(3,5)P2 to LBS2 and PA to LBS3. Thus, targeting the N-terminal lipid binding sites of ATP13A2 might offer a therapeutic approach to reduce cellular toxicity of various PD insults including mitochondrial stress. PMID:27073711

  8. Proposed carrier lipid-binding site of undecaprenyl pyrophosphate phosphatase from Escherichia coli.

    PubMed

    Chang, Hsin-Yang; Chou, Chia-Cheng; Hsu, Min-Feng; Wang, Andrew H J

    2014-07-01

    Undecaprenyl pyrophosphate phosphatase (UppP), an integral membrane protein, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in the bacterial cell wall synthesis. Sequence alignment reveals two consensus regions, containing glutamate-rich (E/Q)XXXE plus PGXSRSXXT motifs and a histidine residue, specific to the bacterial UppP enzymes. The predicted topological model suggests that both of these regions are localized near the aqueous interface of UppP and face the periplasm, implicating that its enzymatic function is on the outer side of the plasma membrane. The mutagenesis analysis demonstrates that most of the mutations (E17A/E21A, H30A, S173A, R174A, and T178A) within the consensus regions are completely inactive, indicating that the catalytic site of UppP is constituted by these two regions. Enzymatic analysis also shows an absolute requirement of magnesium or calcium ions in enzyme activity. The three-dimensional structural model and molecular dynamics simulation studies have shown a plausible structure of the catalytic site of UppP and thus provides insights into the molecular basis of the enzyme-substrate interaction in membrane bilayers. PMID:24855653

  9. Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid.

    PubMed

    Stanislawski, L; Courtney, H S; Simpson, W A; Hasty, D L; Beachey, E H; Robert, L; Ofek, I

    1987-08-01

    In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain. PMID:3298457

  10. Lipid binding capacity of spider hemocyanin.

    PubMed

    Cunningham, M; Gómez, C; Pollero, R

    1999-09-01

    The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999. PMID:10451413

  11. The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

    PubMed Central

    Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

    2013-01-01

    Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

  12. NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

    PubMed

    Yang, Yinshan; Boze, Hélène; Chemardin, Patrick; Padilla, André; Moulin, Guy; Tassanakajon, Anchalee; Pugnière, Martine; Roquet, Françoise; Destoumieux-Garzón, Delphine; Gueguen, Yannick; Bachère, Evelyne; Aumelas, André

    2009-03-01

    The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs. PMID:19107926

  13. Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites

    SciTech Connect

    Andorn, A.C.; Bacon, B.R.; Nguyen-Hunh, A.T.; Parlato, S.J.; Stitts, J.A.

    1988-02-01

    The human caudate and putamen contain two high affinity binding sites for (/sup 3/H)spiroperidol. Both of these affinity states exhibit dopaminergic selectivity. Ascorbic acid, at 0.1 mM, induces a slow loss of the low affinity component of (/sup 3/H)spiroperidol binding in these tissues. The addition of guanyl nucleotides to the ascorbate produces a more rapid loss of (/sup 3/H)spiroperidol binding which includes a loss of the highest affinity state for (/sup 3/H)spiroperidol. Ascorbate induces lipid peroxidation in human caudate and putamen, an effect that is further enhanced by guanyl and inosine nucleotides. In the absence of ascorbate, guanyl nucleotides have no effect on (/sup 3/H)spiroperidol binding but do decrease the affinity of dopamine at each affinity state greater than 60-fold. In the absence of ascorbate, guanyl nucleotides apparently decrease agonist affinity at human brain dopamine2-binding sites without causing an interconversion of agonist affinity states.

  14. Solution Structure of a Repeated Unit of the ABA-1 Nematode Polyprotein Allergen of Ascaris Reveals a Novel Fold and Two Discrete Lipid-Binding Sites

    PubMed Central

    Meenan, Nicola A. G.; Ball, Graeme; Bromek, Krystyna; Uhrín, Dušan; Cooper, Alan; Kennedy, Malcolm W.; Smith, Brian O.

    2011-01-01

    Background Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control. Methodology/Principal Findings The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites. Conclusions/Significance This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or

  15. Crystal Structure of Phosphatidylglycerophosphatase (PGPase), a Putative Membrane-Bound Lipid Phosphatase, Reveals a Novel Binuclear Metal Binding Site and Two Proton Wires

    SciTech Connect

    Kumaran,D.; Bonnano, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 Angstroms resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca{sup 2+} and Mg{sup 2+}, forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of this enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed 'proton wires', is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.

  16. A polyether biotoxin binding site on the lipid-exposed face of the pore domain of Kv channels revealed by the marine toxin gambierol

    PubMed Central

    Kopljar, Ivan; Labro, Alain J.; Cuypers, Eva; Johnson, Henry W. B.; Rainier, Jon D.; Tytgat, Jan; Snyders, Dirk J.

    2009-01-01

    Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC50 of 1.2 ± 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 μM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1–Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general. PMID:19482941

  17. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  18. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    NASA Technical Reports Server (NTRS)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  19. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    PubMed

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-01

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. PMID:27015007

  20. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  1. LIPID - BINDING SURFACES OF MEMBRANE PROTEINS: EVIDENCE FROM EVOLUTIONARY AND STRUCTURAL ANALYSIS

    PubMed Central

    Adamian, Larisa; Naveed, Hammad; Liang, Jie

    2011-01-01

    Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles. In this study, we have assessed the evolutionary selection pressure experienced at lipid-binding sites in a set of α-helical and β-barrel membrane proteins using posterior probability analysis of the ratio of synonymous vs. nonsynonymous substitutions (ω-ratio). We have also carried out a geometric analysis of the membrane protein structures to identify residues in close contact with co-crystallized lipids. We found that residues forming cholesterol-binding sites in both β2-adrenergic receptor and Na+-K+-ATPase exhibit strong conservation, which can be characterized by an expanded cholesterol consensus motif for GPCRs. Our results suggest the functional importance of aromatic stacking interactions and interhelical hydrogen bonds in facilitating protein-cholesterol interactions, which is now reflected in the expanded motif. We also find that residues forming the cardiolipin-binding site in formate dehydrogenase-N γ-subunit and the phosphatidylglycerol binding site in KcsA are under strong purifying selection pressure. Although the lipopolysaccharide (LPS)-binding site in ferric hydroxamate uptake receptor (FhuA) is only weakly conserved, we show using a statistical mechanical model that LPS binds to the least stable FhuA β-strand and protects it from the bulk lipid. Our results suggest that specific lipid binding may be a general mechanism employed by β-barrel membrane proteins to stabilize weakly stable regions. Overall, we find that the residues forming specific lipid binding sites on the surfaces of membrane proteins often experience strong purifying selection pressure. PMID:21167813

  2. Spastin Binds to Lipid Droplets and Affects Lipid Metabolism

    PubMed Central

    Papadopoulos, Chrisovalantis; Orso, Genny; Mancuso, Giuseppe; Herholz, Marija; Gumeni, Sentiljana; Tadepalle, Nimesha; Jüngst, Christian; Tzschichholz, Anne; Schauss, Astrid; Höning, Stefan; Trifunovic, Aleksandra; Daga, Andrea; Rugarli, Elena I.

    2015-01-01

    Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP. PMID:25875445

  3. Bilirubin Binding to PPARα Inhibits Lipid Accumulation.

    PubMed

    Stec, David E; John, Kezia; Trabbic, Christopher J; Luniwal, Amarjit; Hankins, Michael W; Baum, Justin; Hinds, Terry D

    2016-01-01

    Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin. PMID:27071062

  4. Bilirubin Binding to PPARα Inhibits Lipid Accumulation

    PubMed Central

    Stec, David E.; John, Kezia; Trabbic, Christopher J.; Luniwal, Amarjit; Hankins, Michael W.; Baum, Justin

    2016-01-01

    Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin. PMID:27071062

  5. Fluorescence Analysis of the Lipid Binding-induced Conformational Change of Apolipoprotein E4†

    PubMed Central

    Mizuguchi, Chiharu; Hata, Mami; Dhanasekaran, Padmaja; Nickel, Margaret; Phillips, Michael C.; Lund-Katz, Sissel; Saito, Hiroyuki

    2012-01-01

    Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipids-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL3 suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL3. PMID:22730894

  6. Role of membrane lipids in peptide hormone function: binding of enkephalins to micelles.

    PubMed Central

    Deber, C M; Behnam, B A

    1984-01-01

    In the course of their biological function, peptide hormones must be transferred from an aqueous phase to the lipid-rich environment of their membrane-bound receptor proteins. We have investigated the possible influence of phospholipids in this process, using 360-MHz 1H and 90-MHz 13C NMR spectroscopy to examine the association of the opioid peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipid was monitored in NMR spectra by selective chemical shift movements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and alpha-carbon resonances). Results established that the zwitterionic hormones associate hydrophobically both with a neutral lipid (lysophosphatidylcholine) and (also electrostatically) with a negative lipid (lysophosphatidylglycerol). An association constant of Ka = 3.7 X 10(1) M-1 was calculated for the hydrophobic binding of enkephalin to lysophosphatidylcholine. NMR data suggested that enkephalin binds to the lipid with Met/Leu, Phe, and likely Tyr side-chain substituents associated with nonpolar interior regions of the micelle, whereas the COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" model is proposed for hormone-lipid association wherein negative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentration, and orientation into the lipid phase. PMID:6320173

  7. Membrane proteins bind lipids selectively to modulate their structure and function

    PubMed Central

    Allison, Timothy M.; Ulmschneider, Martin B.; Degiacomi, Matteo T.; Baldwin, Andrew J.; Robinson, Carol V.

    2014-01-01

    Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these

  8. Binding sites associated with inhibition of photosystem II

    SciTech Connect

    Shipman, L.L.

    1981-01-01

    A variety of experimental and theoretical evidence has been integrated into coherent molecular mechanisms for the action of photosystem II herbicides. Photosystem II herbicides act by inhibiting electron transfers between the first and second plastoquinones on the reducing side of photosystem II. Each herbicide molecule must have a flat polar component with hydrophobic substituents to be active. The hydrophobic substituents serve to partition the molecule into lipid regions of the cell and to fit the hydrophobic region of the herbicide binding site. The flat polar portion of the herbicide is used for electrostatic binding to the polar region of the herbicide binding site. Theoretical calculations have been carried out to investigate the binding of herbicides to model proteinaceous binding sites.

  9. Identification of lipids and lipid-binding proteins in phloem exudates from Arabidopsis thaliana

    PubMed Central

    Guelette, Brandon S.; Benning, Urs F.; Hoffmann-Benning, Susanne

    2012-01-01

    The phloem plays a crucial role in assimilate and nutrient transport, pathogen response, and plant growth and development. Yet, few species have yielded pure phloem exudate and, if proteins need to be analysed, those species may not have sequenced genomes, making identification difficult. The enrichment of Arabidopsis thaliana phloem exudate in amounts large enough to allow for metabolite and protein analysis is described. Using this method, it was possible to identify 65 proteins present in the Arabidopsis phloem exudate. The majority of these proteins could be grouped by response to pathogens, stress, or hormones, carbon metabolism, protein interaction, modification, and turnover, and transcription factors. It was also possible to detect 11 proteins that play a role in lipid/fatty acid metabolism (aspartic protease, putative 3-β-hydroxysteroid dehydrogenase, UDP-sulphoquinovose synthase/SQD1, lipase, PIG-P-like protein: phosphatidylinositol-N-acetylglucosaminyltransferase), storage (glycine-rich protein), binding (annexin, lipid-associated family protein, GRP17/oleosin), and/or signalling (annexin, putative lipase, PIG-P-like protein). Along with putative lipid-binding proteins, several lipids and fatty acids could be identified. Only a few examples exist of lipids (jasmonic acid, oxylipins) or lipid-binding proteins (DIR1, acyl-CoA-binding protein) in the phloem. Finding hydrophobic compounds in an aqueous environment is not without precedence in biological systems: human blood contains a variety of lipids, many of which play a significant role in human health. In blood, lipids are transported while bound to proteins. The present findings of lipids and lipid-binding proteins in phloem exudates suggest that a similar long-distance lipid signalling exists in plants and may play an important role in plant growth and development. PMID:22442409

  10. Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

    PubMed

    Le Roux, Anabel-Lise; Busquets, Maria Antònia; Sagués, Francesc; Pons, Miquel

    2016-02-01

    Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface. PMID:26638178

  11. Structural Analysis of Papain-Like NlpC/P60 Superfamily Enzymes with a Circularly Permuted Topology Reveals Potential Lipid Binding Sites

    SciTech Connect

    Xu, Qingping; Rawlings, Neil D.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-Andre; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2012-07-11

    NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, 'closed' conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.

  12. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site. [R

    SciTech Connect

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  13. New Insights into Nisin's Antibacterial Mechanism Revealed by Binding Studies with Synthetic Lipid II Analogues.

    PubMed

    't Hart, Peter; Oppedijk, Sabine F; Breukink, Eefjan; Martin, Nathaniel I

    2016-01-12

    Nisin is the preeminent lantibiotic, and to date its antibacterial mechanism has been investigated using a variety of techniques. While nisin's lipid II-mediated mode of action is well-established, a detailed analysis of the thermodynamic parameters governing this interaction has not been previously reported. We here describe an approach employing isothermal titration calorimetry to directly measure the affinity of nisin for lipid II and a number of synthetic lipid II precursors and analogues. Our measurements confirm the pyrophosphate unit of lipid II as the primary site of nisin binding and also indicate that the complete MurNAc moiety is required for a high-affinity interaction. Additionally, we find that while the pentapeptide unit of the lipid II molecule is not required for strong binding by nisin, it does play an important role in stabilizing the subsequently formed nisin-lipid II pore complex, albeit at an entropic cost. The anchoring of lipid II in a membrane environment was also found to play a significant role in enhancing nisin binding and is required in order to achieve a high-affinity interaction. PMID:26653142

  14. Receptor-binding sites: bioinformatic approaches.

    PubMed

    Flower, Darren R

    2006-01-01

    It is increasingly clear that both transient and long-lasting interactions between biomacromolecules and their molecular partners are the most fundamental of all biological mechanisms and lie at the conceptual heart of protein function. In particular, the protein-binding site is the most fascinating and important mechanistic arbiter of protein function. In this review, I examine the nature of protein-binding sites found in both ligand-binding receptors and substrate-binding enzymes. I highlight two important concepts underlying the identification and analysis of binding sites. The first is based on knowledge: when one knows the location of a binding site in one protein, one can "inherit" the site from one protein to another. The second approach involves the a priori prediction of a binding site from a sequence or a structure. The full and complete analysis of binding sites will necessarily involve the full range of informatic techniques ranging from sequence-based bioinformatic analysis through structural bioinformatics to computational chemistry and molecular physics. Integration of both diverse experimental and diverse theoretical approaches is thus a mandatory requirement in the evaluation of binding sites and the binding events that occur within them. PMID:16671408

  15. Binding Orientations and Lipid Interactions of Human Amylin at Zwitterionic and Anionic Lipid Bilayers

    PubMed Central

    Qian, Zhenyu; Jia, Yan; Wei, Guanghong

    2016-01-01

    Increasing evidence suggests that the interaction of human islet amyloid polypeptide (hIAPP) with lipids may facilitate hIAPP aggregation and cause the death of pancreatic islet β-cells. However, the detailed hIAPP-membrane interactions and the influences of lipid compositions are unclear. In this study, as a first step to understand the mechanism of membrane-mediated hIAPP aggregation, we investigate the binding behaviors of hIAPP monomer at zwitterionic palmitoyloleoyl-phosphatidylcholine (POPC) bilayer by performing atomistic molecular dynamics simulations. The results are compared with those of hIAPP at anionic palmitoyloleoyl-phosphatidylglycerol (POPG) bilayers. We find that the adsorption of hIAPP to POPC bilayer is mainly initiated from the C-terminal region and the peptide adopts a helical structure with multiple binding orientations, while the adsorption to POPG bilayer is mostly initiated from the N-terminal region and hIAPP displays one preferential binding orientation, with its hydrophobic residues exposed to water. hIAPP monomer inserts into POPC lipid bilayers more readily than into POPG bilayers. Peptide-lipid interaction analyses show that the different binding features of hIAPP at POPC and POPG bilayers are attributed to different magnitudes of electrostatic and hydrogen-bonding interactions with lipids. This study provides mechanistic insights into the different interaction behaviors of hIAPP with zwitterionic and anionic lipid bilayers. PMID:26649316

  16. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  17. Structural changes in human tear lipocalins associated with lipid binding.

    PubMed

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

    1998-07-28

    Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp17, is buried in both holo- and apo-TL. The steady state exposure of Trp17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface. PMID:9675263

  18. The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport.

    PubMed

    Alva, Vikram; Lupas, Andrei N

    2016-08-01

    The tubular lipid-binding (TULIP) superfamily has emerged in recent years as a major mediator of lipid sensing and transport in eukaryotes. It currently encompasses three protein families, SMP-like, BPI-like, and Takeout-like, which share a common fold. This fold consists of a long helix wrapped in a highly curved anti-parallel β-sheet, enclosing a central, lipophilic cavity. The SMP-like proteins, which include subunits of the ERMES complex and the extended synaptotagmins (E-Syts), appear to be mainly located at membrane contacts sites (MCSs) between organelles, mediating inter-organelle lipid exchange. The BPI-like proteins, which include the bactericidal/permeability-increasing protein (BPI), the LPS (lipopolysaccharide)-binding protein (LBP), the cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP), are either involved in innate immunity against bacteria through their ability to sense lipopolysaccharides, as is the case for BPI and LBP, or in lipid exchange between lipoprotein particles, as is the case for CETP and PLTP. The Takeout-like proteins, which are comprised of insect juvenile hormone-binding proteins and arthropod allergens, transport, where known, lipid hormones to target tissues during insect development. In all cases, the activity of these proteins is underpinned by their ability to bind large, hydrophobic ligands in their central cavity and segregate them away from the aqueous environment. Furthermore, where they are involved in lipid exchange, recent structural studies have highlighted their ability to establish lipophilic, tubular channels, either between organelles in the case of SMP domains or between lipoprotein particles in the case of CETP. Here, we review the current knowledge on the structure, versatile functions, and evolution of the TULIP superfamily. We propose a deep evolutionary split in this superfamily, predating the Last Eukaryotic Common Ancestor, between the SMP-like proteins, which act on

  19. Inhibition of cell-cell binding by lipid assemblies

    DOEpatents

    Nagy, Jon O.; Bargatze, Robert F.

    2001-05-22

    This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

  20. (/sup 3/)tetrahydrotrazodone binding. Association with serotonin binding sites

    SciTech Connect

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-05-01

    High (17 nM) and low (603 nM) affinity binding sites for (/sup 3/)tetrahydrotrazodone ((/sup 3/) THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of (/sup 3/)THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, (/sup 3/) THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that (/sup 3/)THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors.

  1. Biogenesis of the multifunctional lipid droplet: Lipids, proteins, and sites

    PubMed Central

    Gross, Steven P.

    2014-01-01

    Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle’s cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis. PMID:24590170

  2. Binding of Hemagglutinin and Influenza Virus to a Peptide-Conjugated Lipid Membrane

    PubMed Central

    Matsubara, Teruhiko; Shibata, Rabi; Sato, Toshinori

    2016-01-01

    Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV. PMID:27092124

  3. Binding of Hemagglutinin and Influenza Virus to a Peptide-Conjugated Lipid Membrane.

    PubMed

    Matsubara, Teruhiko; Shibata, Rabi; Sato, Toshinori

    2016-01-01

    Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV. PMID:27092124

  4. Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function.

    PubMed

    Watkins, E B; Miller, C E; Majewski, J; Kuhl, T L

    2011-04-26

    Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane's properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin. PMID:21474780

  5. Ethylene binding site affinity in ripening apples

    SciTech Connect

    Blankenship, S.M. . Dept. of Horticultural Science); Sisler, E.C. )

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  6. Reversible calcitonin binding to solubilized sheep brain binding sites.

    PubMed Central

    Sexton, P M; Schneider, H G; D'Santos, C S; Mendelsohn, F A; Kemp, B E; Moseley, J M; Martin, T J; Findlay, D M

    1991-01-01

    In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125I-labelled salmon CT (125I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with beta-octyl glucoside, 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +/- 0.5 nM and a maximum binding site concentration of 6.2 +/- 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems, Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4 degree C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11,18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr approximately solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified. one of Mr approximately 71,000 and another of Mr approximately 88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues. Images Fig. 1. Fig. 6. PMID:1846527

  7. Localization of the chaperone binding site

    NASA Technical Reports Server (NTRS)

    Boyle, D.; Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

  8. Muscarine binding sites in bovine adrenal medulla.

    PubMed

    Barron, B A; Murrin, L C; Hexum, T D

    1986-03-18

    The presence of muscarinic binding sites in the bovine adrenal medulla was investigated using [3H]QNB and the bovine adrenal medulla. Scatchard analysis combined with computer analysis yielded data consistent with a two binding site configuration. KDs of 0.15 and 14 nM and Bmax s of 29 and 210 fmol/mg protein, respectively, were observed. Displacement of [3H]QNB by various cholinergic agents is, in order of decreasing potency: QNB, dexetimide, atropine, scopolamine, imipramine, desipramine, oxotremorine, pilocarpine, acetylcholine, methacholine and carbachol. These results demonstrate the presence of more than one muscarine binding site in the bovine adrenal gland. PMID:3709656

  9. Multiple instance learning of Calmodulin binding sites

    PubMed Central

    Minhas, Fayyaz ul Amir Afsar; Ben-Hur, Asa

    2012-01-01

    Motivation: Calmodulin (CaM) is a ubiquitously conserved protein that acts as a calcium sensor, and interacts with a large number of proteins. Detection of CaM binding proteins and their interaction sites experimentally requires a significant effort, so accurate methods for their prediction are important. Results: We present a novel algorithm (MI-1 SVM) for binding site prediction and evaluate its performance on a set of CaM-binding proteins extracted from the Calmodulin Target Database. Our approach directly models the problem of binding site prediction as a large-margin classification problem, and is able to take into account uncertainty in binding site location. We show that the proposed algorithm performs better than the standard SVM formulation, and illustrate its ability to recover known CaM binding motifs. A highly accurate cascaded classification approach using the proposed binding site prediction method to predict CaM binding proteins in Arabidopsis thaliana is also presented. Availability: Matlab code for training MI-1 SVM and the cascaded classification approach is available on request. Contact: fayyazafsar@gmail.com or asa@cs.colostate.edu PMID:22962461

  10. Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

    PubMed Central

    Konshina, Anastasia G.; Boldyrev, Ivan A.; Utkin, Yuri N.; Omel'kov, Anton V.; Efremov, Roman G.

    2011-01-01

    The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of

  11. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    PubMed Central

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  12. Polyphosphoinositide binding domains: key to inositol lipid biology

    PubMed Central

    Hammond, Gerald R. V.; Balla, Tamas

    2014-01-01

    Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions. In this review, we discuss current knowledge of the principle types of PPIn-protein interactions, focusing on specific lipid-binding domains. We then discuss how these domains have been re-tasked by biologists as molecular probes for these lipids in living cells. Finally, we describe how the knowledge gained with these probes, when combined with other techniques, has led to the current view of the lipids’ localization and function in eukaryotes, focusing mainly on animal cells. PMID:25732852

  13. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  14. Probing the binding of cationic lipids with dendrimers.

    PubMed

    Mandeville, J S; Bourassa, P; Tajmir-Riahi, H A

    2013-01-14

    Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. We report the bindings of several lipids cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), dioctadecyldimethylammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to dendrimers of different compositions such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) under physiological conditions. FTIR, UV-visible spectroscopic, methods and molecular modeling were used to analyze the lipid binding mode, the binding constant, and the effects of lipid complexation on the dendrimer structure. The structural analysis showed that lipids bind dendrimers through both hydrophilic and hydrophobic contacts with overall binding constants of K(chol-mPEG-G3) = 1.7 × 10(3) M(-1), K(chol-mPEG-PAMAM-G4) = 2.7 × 10(3) M(-1), K(chol-PAMAM-G4) = 1.0 × 10(3) M(-1), K(DOPE-mPEG-G3) = 1.5 × 10(3) M(-1), K(DOPE-mPEG-PAMAM-G4) = 1.6 × 10(3) M(-1), K(DOPE-PAMAM-G4) = 5.3 × 10(2) M(-1), K(DDAB-mPEG-G3) = 1.5 × 10(3) M(-1), K(DDAB-mPEG-PAMAM-G4) = 1.9 × 10(2) M(-1), K(DDAB-PAMAM-G4) = 7.0 × 10(2) M(-1), K(DOTAP-mPEG-G3) = 1.9 × 10(3) M(-1), K(DOTAP-mPEG-PAMAM-G4) = 1.5 × 10(3) M(-1), and K(DOTAP-PAMAM-G4) = 5.7 × 10(2) M(-1). Weaker interaction was observed as dendrimer cationic charges increased. The free binding energies from docking were -5.15 (cholesterol), -5.79 (DDAB), and -5.36 kcal/mol (DOTAP) with the order of stability DDAB-PAMAM-G-4 > DOTAP-PAMAM-G4 > cholesterol-PAMAM-G4, consistent with the spectroscopic results. Dendrimers might act as carriers to transport lipids in vitro. PMID:23130659

  15. 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

  16. OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites.

    PubMed

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

    2016-01-01

    Oxysterol-binding protein (OSBP) and its related protein homologs, ORPs, constitute a conserved family of lipid-binding/transfer proteins (LTPs) expressed ubiquitously in eukaryotes. The ligand-binding domain of ORPs accommodates cholesterol and oxysterols, but also glycerophospholipids, particularly phosphatidylinositol-4-phosphate (PI4P). ORPs have been implicated as intracellular lipid sensors or transporters. Most ORPs carry targeting determinants for the endoplasmic reticulum (ER) and non-ER organelle membrane. ORPs are located and function at membrane contact sites (MCSs), at which ER is closely apposed with other organelle limiting membranes. Such sites have roles in lipid transport and metabolism, control of Ca(2+) fluxes, and signaling events. ORPs are postulated either to transport lipids over MCSs to maintain the distinct lipid compositions of organelle membranes, or to control the activity of enzymes/protein complexes with functions in signaling and lipid metabolism. ORPs may transfer PI4P and another lipid class bidirectionally. Transport of PI4P followed by its hydrolysis would in this model provide the energy for transfer of the other lipid against its concentration gradient. Control of organelle lipid compositions by OSBP/ORPs is important for the life cycles of several pathogenic viruses. Targeting ORPs with small-molecular antagonists is proposed as a new strategy to combat viral infections. Several ORPs are reported to modulate vesicle transport along the secretory or endocytic pathways. Moreover, antagonists of certain ORPs inhibit cancer cell proliferation. Thus, ORPs are LTPs, which mediate interorganelle lipid transport and coordinate lipid signals with a variety of cellular regimes. PMID:26811291

  17. Identification of consensus binding sites clarifies FMRP binding determinants.

    PubMed

    Anderson, Bart R; Chopra, Pankaj; Suhl, Joshua A; Warren, Stephen T; Bassell, Gary J

    2016-08-19

    Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function. PMID:27378784

  18. Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes.

    PubMed

    Tatulian, S A

    1983-12-21

    Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (sigma max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol greater than ClO-4 greater than I- greater than SCN- greater than Br- greater than NO-3 greater than Cl- approximately or equal to SO2-4. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of sigma max. The binding sites were considered as defects in the package of lipid molecules in membranes. PMID:6652082

  19. Receptor-like function of heparin in the binding and uptake of neutral lipids

    SciTech Connect

    Bosner, M.S.; Gulick, T.; Riley, D.J.S.; Spilburg, C.A.; Lange, L.G. III )

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, the authors postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic {sup 125}I-labeled cholesterol esterase and {sup 125}I-labeled triglyceride lipase. This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a K{sub d} of 100 nM and a B{sub max} of approximately 50-60 pmol per mg of vesicle protein. They conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalized pathway for transport of neutral lipids into endothelial and other cells.

  20. Nonspecific Binding Domains in Lipid Membranes Induced by Phospholipase A2.

    PubMed

    Hong, Chia Yee; Han, Chung-Ta; Chao, Ling

    2016-07-12

    Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and is thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support, and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind. PMID:27218880

  1. The binding of Maize DHN1 to Lipid Vesicles. Gain of Structure and Lipid Specificity1

    PubMed Central

    Koag, Myong-Chul; Fenton, Raymond D.; Wilkens, Stephan; Close, Timothy J.

    2003-01-01

    Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to abiotic stresses such as drought, low temperature, and salinity or during the late stages of embryogenesis. Spectral and thermal properties of these proteins in purified form suggest that they are “intrinsically unstructured.” However, DHNs contain at least one copy of a consensus 15-amino acid sequence, the “K segment,” which resembles a class A2 amphipathic α-helical, lipid-binding domain found in other proteins such as apolipoproteins and α-synuclein. The presence of the K segment raises the question of whether DHNs bind lipids, bilayers, or phospholipid vesicles. Here, we show that maize (Zea mays) DHN DHN1 can bind to lipid vesicles that contain acidic phospholipids. We also observe that DHN1 binds more favorably to vesicles of smaller diameter than to larger vesicles, and that the association of DHN1 with vesicles results in an apparent increase of α-helicity of the protein. Therefore, DHNs, and presumably somewhat similar plant stress proteins in the late embryogenesis abundant and cold-regulated classes may undergo function-related conformational changes at the water/membrane interface, perhaps related to the stabilization of vesicles or other endomembrane structures under stress conditions. PMID:12529538

  2. Predicting Ca(2+)-binding sites in proteins.

    PubMed Central

    Nayal, M; Di Cera, E

    1994-01-01

    The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins. Images Fig. 4 PMID:8290605

  3. Receptor-Like Function of Heparin in the Binding and Uptake of Neutral Lipids

    NASA Astrophysics Data System (ADS)

    Bosner, Matthew S.; Gulick, Tod; Riley, D. J. S.; Spilburg, Curtis A.; Lange, Louis G.

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.

  4. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

    PubMed

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G; Módos, Károly; Kardos, József; Liliom, Károly

    2010-10-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well. PMID:20522785

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

    PubMed Central

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

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions. PMID:26540053

  6. Radiation inactivation reveals discrete cation binding sites that modulate dihydropyridine binding sites

    SciTech Connect

    Bolger, G.T.; Skolnick, P.; Kempner, E.S. )

    1989-08-01

    In low ionic strength buffer (5 mM Tris.HCl), the binding of (3H) nitrendipine to dihydropyridine calcium antagonist binding sites of mouse forebrain membranes is increased by both Na{sup +} and Ca{sup 2+}. Radiation inactivation was used to determine the target size of ({sup 3}H)nitrendipine binding sites in 5 mM Tris.HCl buffer, in the presence and absence of these cations. After irradiation, ({sup 3}H) nitrendipine binding in buffer with or without Na+ was diminished, due to a loss of binding sites and also to an increase in Kd. After accounting for radiation effects on the dissociation constant, the target size for the nitrendipine binding site in buffer was 160-170 kDa and was 170-180 kDa in the presence of sodium. In the presence of calcium ions, ({sup 3}H)nitrendipine binding showed no radiation effects on Kd and yielded a target size of 150-170 kDa. These findings suggest, as in the case of opioid receptors, the presence of high molecular weight membrane components that modulate cation-induced alterations in radioligand binding to dihydropyridine binding sites.

  7. Dynamics of Transcription Factor Binding Site Evolution

    PubMed Central

    Tuğrul, Murat; Paixão, Tiago; Barton, Nicholas H.; Tkačik, Gašper

    2015-01-01

    Evolution of gene regulation is crucial for our understanding of the phenotypic differences between species, populations and individuals. Sequence-specific binding of transcription factors to the regulatory regions on the DNA is a key regulatory mechanism that determines gene expression and hence heritable phenotypic variation. We use a biophysical model for directional selection on gene expression to estimate the rates of gain and loss of transcription factor binding sites (TFBS) in finite populations under both point and insertion/deletion mutations. Our results show that these rates are typically slow for a single TFBS in an isolated DNA region, unless the selection is extremely strong. These rates decrease drastically with increasing TFBS length or increasingly specific protein-DNA interactions, making the evolution of sites longer than ∼ 10 bp unlikely on typical eukaryotic speciation timescales. Similarly, evolution converges to the stationary distribution of binding sequences very slowly, making the equilibrium assumption questionable. The availability of longer regulatory sequences in which multiple binding sites can evolve simultaneously, the presence of “pre-sites” or partially decayed old sites in the initial sequence, and biophysical cooperativity between transcription factors, can all facilitate gain of TFBS and reconcile theoretical calculations with timescales inferred from comparative genomics. PMID:26545200

  8. Preferred Metal Binding Site of Aniline

    NASA Astrophysics Data System (ADS)

    Kumari, Sudesh; Sohnlein, Brad; Yang, Dong-Sheng

    2012-06-01

    Group III metal-aniline complexes, M-aniline (M = Sc, Y, and La), were produced by interactions between laser-vaporized metal atoms and aniline vapor in a pulsed molecular beam source, identified by photoionization time-of-flight mass spectrometry, and studied by pulsed-field ionization zero electron kinetic energy (ZEKE) spectroscopy and density functional theory calculations. Adiabatic ionization energies and several vibrational intervals were measured from the ZEKE spectra. Metal binding sites and electronic states were determined by combining the ZEKE measurements and theoretical calculations. Although aniline has various possible sites for metal coordination, the preferred site was determined to be phenyl ring. The metal binding with the phenyl ring yields syn and anti conformers. In these conformers, the neutral complexes are in doublet ground states and the corresponding singly charged cations in singlet states.

  9. 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

  10. Extra-helical binding site of a glucagon receptor antagonist.

    PubMed

    Jazayeri, Ali; Doré, Andrew S; Lamb, Daniel; Krishnamurthy, Harini; Southall, Stacey M; Baig, Asma H; Bortolato, Andrea; Koglin, Markus; Robertson, Nathan J; Errey, James C; Andrews, Stephen P; Teobald, Iryna; Brown, Alastair J H; Cooke, Robert M; Weir, Malcolm; Marshall, Fiona H

    2016-05-12

    Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors. PMID:27111510

  11. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-12-31

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  12. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-01-01

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  13. Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family.

    PubMed

    Tong, Junsen; Manik, Mohammad Kawsar; Yang, Huiseon; Im, Young Jun

    2016-08-01

    Sterols such as cholesterol in mammals and ergosterol in fungi are essential membrane components and play a key role in membrane function and in cell signaling. The intracellular distribution and processing of sterols and other phospholipids are in part carried out by oxysterol binding protein-related proteins (ORPs) in eukaryotes. Seven ORPs (Osh1-Osh7 proteins) in yeast have distinct functions in maintaining distribution, metabolism and signaling of intracellular lipids but they share at least one essential function. Significant progress has been made in understanding the ligand specificity and mechanism of non-vesicular lipid transport by ORPs. The unique structural features of Osh proteins explain the diversity and specificity of functions in PI(4)P-coupled lipid transport optimized in membrane contact sites. This review discusses the current advances in structural biology regarding this protein family and its potential functions, introducing them as the key players in the novel pathways of phosphoinositide-coupled directional transport of various lipids. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26784528

  14. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis.

    PubMed

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E; Rigby, Neil M; Mackie, Alan R; Dhaliwal, Balvinder; Mills, E N Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39-40, 56-57 and 79-80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  15. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis

    PubMed Central

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E.; Rigby, Neil M.; Mackie, Alan R.; Dhaliwal, Balvinder; Mills, E. N. Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  16. The prediction of novel multiple lipid-binding regions in protein translocation motor proteins: a possible general feature.

    PubMed

    Keller, Rob C A

    2011-03-01

    Protein translocation is an important cellular process. SecA is an essential protein component in the Sec system, as it contains the molecular motor that facilitates protein translocation. In this study, a bioinformatics approach was applied in the search for possible lipid-binding helix regions in protein translocation motor proteins. Novel lipid-binding regions in Escherichia coli SecA were identified. Remarkably, multiple lipid-binding sites were also identified in other motor proteins such as BiP, which is involved in ER protein translocation. The prokaryotic signal recognition particle receptor FtsY, though not a motor protein, is in many ways related to SecA, and was therefore included in this study. The results demonstrate a possible general feature for motor proteins involved in protein translocation. PMID:20957445

  17. Different modes of lipid binding to membrane proteins probed by mass spectrometry.

    PubMed

    Bechara, Chérine; Robinson, Carol V

    2015-04-29

    The realization that the lipid environment is crucial for maintaining the structure and function of membrane proteins prompts new methods to understand lipid interactions. One such method, mass spectrometry, is emerging with the potential to monitor different modes of lipid binding to membrane protein complexes. Initial studies monitored the addition of lipids and deduced the kinetic and thermodynamic effects of lipid binding to proteins. Recent efforts however have focused on identifying lipids already present, explicitly in plugs, annular rings, or cavities. Lipids that bind within these orifices to membrane proteins will have higher residence times than those in the bulk lipid bilayer and consequently can be quantified and characterized by mass spectrometry. In special cases, lipids identified within cavities have been proposed as substrates following activity assays. Alternatively, a gas-phase unfolding protocol can be used to distinguish lipids that are important for stability. These lipids can subsequently be added during crystallization for the characterization of lipid-bound protein complexes. Overall therefore this Perspective provides an overview of recent advances in mass spectrometry, with a particular focus on the distinction of the various modes of lipid binding, and their implications for structure and function as well as new directions that lie ahead. PMID:25860341

  18. Molecular anatomy of the antibody binding site.

    PubMed

    Novotný, J; Bruccoleri, R; Newell, J; Murphy, D; Haber, E; Karplus, M

    1983-12-10

    The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity. PMID:6643494

  19. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    SciTech Connect

    Rozovsky, Sharon; Forstner, Martin B.; Sondermann, Holger; Groves, Jay T.

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  20. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  1. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    PubMed

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off. PMID:26260520

  2. Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

    PubMed

    Yu, Haijia; Liu, Yinghui; Gulbranson, Daniel R; Paine, Alex; Rathore, Shailendra S; Shen, Jingshi

    2016-04-19

    Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation. PMID:27044075

  3. Oxytocin binding sites in bovine mammary tissue

    SciTech Connect

    Zhao, Xin.

    1989-01-01

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

  4. The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

    PubMed Central

    Grippa, Alexandra; Buxó, Laura; Mora, Gabriel; Funaya, Charlotta; Idrissi, Fatima-Zahra; Mancuso, Francesco; Gomez, Raul; Muntanyà, Júlia; Sabidó, Eduard

    2015-01-01

    Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites. PMID:26572621

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

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-01-01

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs. PMID:18305831

  6. Binding Sites Analyser (BiSA): Software for Genomic Binding Sites Archiving and Overlap Analysis

    PubMed Central

    Khushi, Matloob; Liddle, Christopher; Clarke, Christine L.; Graham, J. Dinny

    2014-01-01

    Genome-wide mapping of transcription factor binding and histone modification reveals complex patterns of interactions. Identifying overlaps in binding patterns by different factors is a major objective of genomic studies, but existing methods to archive large numbers of datasets in a personalised database lack sophistication and utility. Therefore we have developed transcription factor DNA binding site analyser software (BiSA), for archiving of binding regions and easy identification of overlap with or proximity to other regions of interest. Analysis results can be restricted by chromosome or base pair overlap between regions or maximum distance between binding peaks. BiSA is capable of reporting overlapping regions that share common base pairs; regions that are nearby; regions that are not overlapping; and average region sizes. BiSA can identify genes located near binding regions of interest, genomic features near a gene or locus of interest and statistical significance of overlapping regions can also be reported. Overlapping results can be visualized as Venn diagrams. A major strength of BiSA is that it is supported by a comprehensive database of publicly available transcription factor binding sites and histone modifications, which can be directly compared to user data. The documentation and source code are available on http://bisa.sourceforge.net PMID:24533055

  7. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  8. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    DOE PAGESBeta

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-04-28

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all

  9. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling.

    PubMed

    Barbaglia, Allison M; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  10. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling

    PubMed Central

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  11. Binding Site Graphs: A New Graph Theoretical Framework for Prediction of Transcription Factor Binding Sites

    PubMed Central

    Reddy, Timothy E; DeLisi, Charles; Shakhnovich, Boris E

    2007-01-01

    Computational prediction of nucleotide binding specificity for transcription factors remains a fundamental and largely unsolved problem. Determination of binding positions is a prerequisite for research in gene regulation, a major mechanism controlling phenotypic diversity. Furthermore, an accurate determination of binding specificities from high-throughput data sources is necessary to realize the full potential of systems biology. Unfortunately, recently performed independent evaluation showed that more than half the predictions from most widely used algorithms are false. We introduce a graph-theoretical framework to describe local sequence similarity as the pair-wise distances between nucleotides in promoter sequences, and hypothesize that densely connected subgraphs are indicative of transcription factor binding sites. Using a well-established sampling algorithm coupled with simple clustering and scoring schemes, we identify sets of closely related nucleotides and test those for known TF binding activity. Using an independent benchmark, we find our algorithm predicts yeast binding motifs considerably better than currently available techniques and without manual curation. Importantly, we reduce the number of false positive predictions in yeast to less than 30%. We also develop a framework to evaluate the statistical significance of our motif predictions. We show that our approach is robust to the choice of input promoters, and thus can be used in the context of predicting binding positions from noisy experimental data. We apply our method to identify binding sites using data from genome scale ChIP–chip experiments. Results from these experiments are publicly available at http://cagt10.bu.edu/BSG. The graphical framework developed here may be useful when combining predictions from numerous computational and experimental measures. Finally, we discuss how our algorithm can be used to improve the sensitivity of computational predictions of transcription factor

  12. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  13. PIP(2)-binding site in Kir channels: definition by multiscale biomolecular simulations.

    PubMed

    Stansfeld, Phillip J; Hopkinson, Richard; Ashcroft, Frances M; Sansom, Mark S P

    2009-11-24

    Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains. PMID:19839652

  14. OSBP-Related Protein Family in Lipid Transport Over Membrane Contact Sites

    PubMed Central

    Olkkonen, Vesa M.

    2015-01-01

    Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling. PMID:26715851

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

    PubMed Central

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

    2015-01-01

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets. PMID:26655740

  16. Viral infection controlled by a calcium-dependent lipid-binding module in ALIX.

    PubMed

    Bissig, Christin; Lenoir, Marc; Velluz, Marie-Claire; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael; Gruenberg, Jean

    2013-05-28

    ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection. PMID:23664863

  17. Driving force of binding of amyloid {beta}-protein to lipid bilayers

    SciTech Connect

    Ikeda, Keisuke; Matsuzaki, Katsumi

    2008-06-06

    Amyloid {beta}-protein (A{beta}) has been reported to interact with a variety of lipid species, although the thermodynamic driving force remains unclear. We investigated the binding of A{beta}s labeled with the dye diethylaminocoumarin (DAC-A{beta}s) to lipid bilayers under various conditions. DAC-A{beta}-(1-40) electrostatically bound to anionic and cationic lipids at acidic and alkaline interfacial pH, respectively. However, at neutral pH, electroneutral A{beta} did not bind to these lipids, indicating little hydrophobic interaction between A{beta}-(1-40) and the acyl chains of lipids. In contrast, DAC-A{beta} associated with glycolipids even under electroneutral conditions. These results suggested that hydrogen-bonding as well as hydrophobic interactions with sugar groups of glycolipids drive the membrane binding of A{beta}-(1-40)

  18. Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.

    PubMed Central

    Ahlers, M; Grainger, D W; Herron, J N; Lim, K; Ringsdorf, H; Salesse, C

    1992-01-01

    Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interestingly, the observed degrees of quenching were nearly independent of the lipid membrane model studied, but directly correlated with the chemical structure of the lipids. In all cases, the antibody recognized and quenched most efficiently a lipid based on dioctadecylamine where fluorescein is attached to the headgroup via a long, flexible hydrophilic spacer. Dipalmitoyl phosphatidylethanolamine containing a fluorescein headgroup demonstrated only partial binding/quenching. Egg phosphatidylethanolamine with a fluorescein headgroup showed no susceptibility to antibody recognition, binding, or quenching. Formation of two-dimensional protein domains upon antibody binding to the fluorescein-lipids in monolayers is also presented. Chemical and physical requirements for these antibody-hapten complexes at membrane surfaces have been discussed in terms of molecular dynamics simulations based on recent crystallographic models for this antibody-hapten complex (Herron et al., 1989. Proteins Struct. Funct. Genet. 5:271-280). Images FIGURE 7 FIGURE 8 PMID:1420916

  19. The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

    PubMed

    Haji-Ghassemi, Omid; Müller-Loennies, Sven; Rodriguez, Teresa; Brade, Lore; Grimmecke, Hans-Dieter; Brade, Helmut; Evans, Stephen V

    2016-05-01

    Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies. PMID:26933033

  20. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs. PMID:26487699

  1. Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

    PubMed Central

    Pérez, Yolanda; Maffei, Mariano; Igea, Ana; Amata, Irene; Gairí, Margarida; Nebreda, Angel R.; Bernadó, Pau; Pons, Miquel

    2013-01-01

    c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation. PMID:23416516

  2. Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization.

    PubMed

    Chinthalapudi, Krishna; Rangarajan, Erumbi S; Brown, David T; Izard, Tina

    2016-08-23

    The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2 Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling. PMID:27503891

  3. Tuning Genetic Clocks Employing DNA Binding Sites

    PubMed Central

    Jayanthi, Shridhar; Del Vecchio, Domitilla

    2012-01-01

    Periodic oscillations play a key role in cell physiology from the cell cycle to circadian clocks. The interplay of positive and negative feedback loops among genes and proteins is ubiquitous in these networks. Often, delays in a negative feedback loop and/or degradation rates are a crucial mechanism to obtain sustained oscillations. How does nature control delays and kinetic rates in feedback networks? Known mechanisms include proper selection of the number of steps composing a feedback loop and alteration of protease activity, respectively. Here, we show that a remarkably simple means to control both delays and effective kinetic rates is the employment of DNA binding sites. We illustrate this design principle on a widely studied activator-repressor clock motif, which is ubiquitous in natural systems. By suitably employing DNA target sites for the activator and/or the repressor, one can switch the clock “on” and “off” and precisely tune its period to a desired value. Our study reveals a design principle to engineer dynamic behavior in biomolecular networks, which may be largely exploited by natural systems and employed for the rational design of synthetic circuits. PMID:22859962

  4. Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.

    PubMed

    Lohman, Danielle C; Forouhar, Farhad; Beebe, Emily T; Stefely, Matthew S; Minogue, Catherine E; Ulbrich, Arne; Stefely, Jonathan A; Sukumar, Shravan; Luna-Sánchez, Marta; Jochem, Adam; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Wang, Huang; Westphall, Michael S; Wrobel, Russell L; Everett, John K; Mitchell, Julie C; López, Luis C; Coon, Joshua J; Tong, Liang; Pagliarini, David J

    2014-11-01

    Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis. PMID:25339443

  5. Mu opioid receptor binding sites in human brain

    SciTech Connect

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand (/sup 3/H)DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of (/sup 3/H)DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas.

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

    SciTech Connect

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

    2007-01-01

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

  7. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

    PubMed

    Singh, Satinder K; Yamashita, Atsuko; Gouaux, Eric

    2007-08-23

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

  8. 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

  9. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes.

    PubMed

    Sezgin, Erdinc; Levental, Ilya; Grzybek, Michal; Schwarzmann, Günter; Mueller, Veronika; Honigmann, Alf; Belov, Vladimir N; Eggeling, Christian; Coskun, Unal; Simons, Kai; Schwille, Petra

    2012-07-01

    Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GMI exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact

  10. Autoradiographic localization of endothelin-1 binding sites in porcine skin

    SciTech Connect

    Zhao, Y.D.; Springall, D.R.; Wharton, J.; Polak, J.M. )

    1991-01-01

    Autoradiographic techniques and {sup 125}I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.

  11. Unraveling determinants of transcription factor binding outside the core binding site.

    PubMed

    Levo, Michal; Zalckvar, Einat; Sharon, Eilon; Dantas Machado, Ana Carolina; Kalma, Yael; Lotam-Pompan, Maya; Weinberger, Adina; Yakhini, Zohar; Rohs, Remo; Segal, Eran

    2015-07-01

    Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites. PMID:25762553

  12. Protein Function Annotation By Local Binding Site Surface Similarity

    PubMed Central

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

    2013-01-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against approximately 60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that pre-dated query protein biochemical annotation for five out of the eight query proteins. A panel of twelve currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins. PMID:24166661

  13. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments. PMID:27410740

  14. A lipid-regulated docking site on vinculin for protein kinase C.

    PubMed

    Ziegler, Wolfgang H; Tigges, Ulrich; Zieseniss, Anke; Jockusch, Brigitte M

    2002-03-01

    During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes. PMID:11741957

  15. Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

    PubMed Central

    Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

    2015-01-01

    SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

  16. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets

    PubMed Central

    Palermo, Giulia; Bauer, Inga; Campomanes, Pablo; Cavalli, Andrea; Armirotti, Andrea; Girotto, Stefania; Rothlisberger, Ursula; De Vivo, Marco

    2015-01-01

    The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts. PMID:26111155

  17. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    PubMed

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  18. The molecular architecture of protein-protein binding sites.

    PubMed

    Reichmann, Dana; Rahat, Ofer; Cohen, Mati; Neuvirth, Hani; Schreiber, Gideon

    2007-02-01

    The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions? PMID:17239579

  19. Sizes of Mn-binding sites in spinach thylakoids

    SciTech Connect

    Takahashi, M.; Asada, K.

    1986-12-25

    The sizes of the Mn-binding sites in spinach thylakoids were estimated by target size analysis, assaying the membrane-bound Mn that was resistant to EDTA washing after radiation inactivation. The inactivation curve showed well the inactivation of two independent Mn-binding sites of different sizes: about two-thirds of the Mn coordinated to a binding site of 65 kDa, and the rest bound to a much smaller site of only about 3 kDa. In the large site, there was about 1 g atom of Mn/110 mol of chlorophyll in spinach thylakoids, which was constant in normally grown plants, although the Mn level in the small site depended on culture conditions. Thylakoids that had been incubated with hydroxylamine or in 0.8 M Tris lost Mn exclusively from the large binding site.

  20. 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

  1. Wnts grasp the WIF domain of Wnt Inhibitory Factor 1 at two distinct binding sites.

    PubMed

    Kerekes, Krisztina; Bányai, László; Patthy, László

    2015-10-01

    Wnts have a structure resembling a hand with "thumb" and "index" fingers that grasp the cysteine rich domains of Frizzled receptors at two distinct binding sites. In the present work we show that the WIF domain of Wnt Inhibitory Factor 1 is also bound by Wnts at two sites. Using C-terminal domains of Wnt5a and Wnt7a and arginine-scanning mutagenesis of the WIF domain we demonstrate that, whereas the N-terminal, lipid-modified "thumb" of Wnts interacts with the alkyl-binding site of the WIF domain, the C-terminal domain of Wnts (Wnt-CTD) binds to a surface on the opposite side of the WIF domain. PMID:26342861

  2. Identification and characterization of anion binding sites in RNA

    SciTech Connect

    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L.

    2010-05-24

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

  3. The Alzheimer's disease Aβ peptide binds to the anionic DMPS lipid bilayer.

    PubMed

    Lockhart, Christopher; Klimov, Dmitri K

    2016-06-01

    We have applied isobaric-isothermal replica exchange molecular dynamics (REMD) and the all-atom explicit water model to study binding of Aβ10-40 peptide to the anionic DMPS bilayer. To provide comparison with a zwitterionic bilayer, we used our previous REMD simulations probing binding of the same peptide to the DMPC bilayer. Using two sets of simulations, we comparatively analyzed the equilibrium Aβ conformational ensemble, peptide-bilayer interactions, and changes in the bilayer structure induced by Aβ binding. Our results are six-fold. (1) Binding to the DMPS bilayer triggers the formation of stable helix in the Aβ C-terminal, although the helix-inducing effect caused by DMPS lipids is weaker than that of DMPC. (2) Compared to the DMPC-bound Aβ monomer, the anionic bilayer weakens intrapeptide interactions, particularly, formed by charged amino acids. (3) Binding of Aβ peptide to the DMPS bilayer is primarily governed by electrostatic interactions between charged amino acids and charged lipid groups. In contrast, these interactions play minor role in Aβ binding to the DMPC bilayer. (4) Aβ peptide generally resides on the DMPS bilayer surface causing relatively minor bilayer thinning. The opposite scenario applies to Aβ binding to the DMPC bilayer. (5) In contrast to DMPC simulations, Aβ largely expels anionic lipids from its binding "footprint" forming a ring of charged amino acids mixed with charged lipid groups around the peptide. (6) Aβ binding disorders proximal DMPS lipids more strongly than their DMPC counterparts. Our simulations show that Aβ monomers fail to perturb anionic or zwitterionic bilayers across both leaflets. PMID:26947182

  4. Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties

    PubMed Central

    Silva-Álvarez, Valeria; Franchini, Gisela R.; Pórfido, Jorge L.; Kennedy, Malcolm W.; Ferreira, Ana M.; Córsico, Betina

    2015-01-01

    Background The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Methodology/Principal Findings Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. Conclusions/Significance We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid

  5. Ab initio prediction of transcription factor binding sites.

    PubMed

    Liu, L Angela; Bader, Joel S

    2007-01-01

    Transcription factors are DNA-binding proteins that control gene transcription by binding specific short DNA sequences. Experiments that identify transcription factor binding sites are often laborious and expensive, and the binding sites of many transcription factors remain unknown. We present a computational scheme to predict the binding sites directly from transcription factor sequence using all-atom molecular simulations. This method is a computational counterpart to recent high-throughput experimental technologies that identify transcription factor binding sites (ChIP-chip and protein-dsDNA binding microarrays). The only requirement of our method is an accurate 3D structural model of a transcription factor-DNA complex. We apply free energy calculations by thermodynamic integration to compute the change in binding energy of the complex due to a single base pair mutation. By calculating the binding free energy differences for all possible single mutations, we construct a position weight matrix for the predicted binding sites that can be directly compared with experimental data. As water-bridged hydrogen bonds between the transcription factor and DNA often contribute to the binding specificity, we include explicit solvent in our simulations. We present successful predictions for the yeast MAT-alpha2 homeodomain and GCN4 bZIP proteins. Water-bridged hydrogen bonds are found to be more prevalent than direct protein-DNA hydrogen bonds at the binding interfaces, indicating why empirical potentials with implicit water may be less successful in predicting binding. Our methodology can be applied to a variety of DNA-binding proteins. PMID:17990512

  6. Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

    PubMed Central

    Münch, Daniela; Engels, Ina; Müller, Anna; Reder-Christ, Katrin; Falkenstein-Paul, Hildegard; Bierbaum, Gabriele; Grein, Fabian; Bendas, Gerd; Sahl, Hans-Georg

    2014-01-01

    Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4′-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit lipid I- and lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor lipid II appears to involve, in addition to the d-Ala-d-Ala terminus, other species-specific binding sites of the lipid II molecule, i.e., the crossbridge and d-isoglutamine in position 2 of the lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified lipid II and modified lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in d-Ala-d-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high

  7. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    SciTech Connect

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  8. Tau Binds to Lipid Membrane Surfaces via Short Amphipathic Helices Located in Its Microtubule-Binding Repeats

    PubMed Central

    Georgieva, Elka R.; Xiao, Shifeng; Borbat, Peter P.; Freed, Jack H.; Eliezer, David

    2014-01-01

    Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer’s disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau’s poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide

  9. 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

  10. Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

    PubMed Central

    Ben-Tal, N; Honig, B; Peitzsch, R M; Denisov, G; McLaughlin, S

    1996-01-01

    We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues. Images FIGURE 2 FIGURE 3 PMID:8842196

  11. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  12. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  13. Temperature and pressure adaptation of the binding site of acetylcholinesterase.

    PubMed

    Hochachka, P W

    1974-12-01

    1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2 degrees C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding. PMID:4462739

  14. Druggability of methyl-lysine binding sites.

    PubMed

    Santiago, C; Nguyen, K; Schapira, M

    2011-12-01

    Structural modules that specifically recognize--or read--methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions. PMID:22146969

  15. Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes

    NASA Astrophysics Data System (ADS)

    Marchini, Cristina; Montani, Maura; Amici, Augusto; Pozzi, Daniela; Caminiti, Ruggero; Caracciolo, Giulio

    2009-01-01

    We have applied electrophoresis on agarose gels to investigate the DNA-binding capacity of cationic liposomes made of cationic DC-cholesterol and neutral dioleoylphosphatidylethanolamine as a function of membrane charge density and cationic lipid/DNA charge ratio. While each cationic liposome formulation exhibits a distinctive DNA-protection ability, here we show that such a capacity is universally regulated by surface area of lipid membranes available for binding in an aspecific manner. The relevance of DNA protection for gene transfection is also discussed.

  16. Tryptophan-binding sites on nuclear envelopes of rat liver

    SciTech Connect

    Kurl, R.; Verney, E.; Sidransky, H.

    1986-03-05

    Tryptophan (TRP), an essential amino acid, has been demonstrated to affect certain cellular processes including transcriptional and translational events in the liver. These events are presumed to be mediated at the nuclear level possibly via binding of TRP to nuclei. In an effort to delineate the role of TRP on these metabolic processes, the nuclear location of these binding sites was investigated. Incubation of isolated, intact, hepatic nuclei with (/sup 3/H)TRP followed by fractionation revealed the presence of about 60% of specific TRP binding to nuclear membranes. This binding reached equilibrium by 2 hours after incubation at room temperature. Scatchard analysis revealed two classes of binding sites: (1) high affinity (K/sub D/ of about 10/sup -10/M) and (2) low affinity (K/sub D/ of about 10/sup -8/M). The inhibition of binding by treatment with either ..beta..-galactosidase or concanavalin A suggested that the binding entity was a glycoprotein. However, treatment with neuraminidase resulted in an increase in binding which suggested that terminal sialic acid residues play a role, possibly an inhibitory one, on TRP binding. The function of these binding sites on the mechanism of TRP action is being investigated.

  17. Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

    SciTech Connect

    Sloan, J.W.

    1984-01-01

    These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) have been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.

  18. Lipid binding promotes oligomerization and focal adhesion activity of vinculin

    PubMed Central

    Chinthalapudi, Krishna; Rangarajan, Erumbi S.; Patil, Dipak N.; George, Eric M.; Brown, David T.

    2014-01-01

    Adherens junctions (AJs) and focal adhesion (FA) complexes are necessary for cell migration and morphogenesis, and for the development, growth, and survival of all metazoans. Vinculin is an essential regulator of both AJs and FAs, where it provides links to the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate (PIP2) affects the functions of many targets, including vinculin. Here we report the crystal structure of vinculin in complex with PIP2, which revealed that PIP2 binding alters vinculin structure to direct higher-order oligomerization and suggests that PIP2 and F-actin binding to vinculin are mutually permissive. Forced expression of PIP2-binding–deficient mutants of vinculin in vinculin-null mouse embryonic fibroblasts revealed that PIP2 binding is necessary for maintaining optimal FAs, for organization of actin stress fibers, and for cell migration and spreading. Finally, photobleaching experiments indicated that PIP2 binding is required for the control of vinculin dynamics and turnover in FAs. Thus, through oligomerization, PIP2 directs a transient vinculin sequestration at FAs that is necessary for proper FA function. PMID:25488920

  19. Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

    PubMed

    Abou-Zied, Osama K

    2015-01-01

    Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena. PMID:25738490

  20. Evidence for a second receptor binding site on human prolactin.

    PubMed

    Goffin, V; Struman, I; Mainfroid, V; Kinet, S; Martial, J A

    1994-12-23

    The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resulting mutants exhibit reduced bioactivity in the Nb2 cell proliferation bioassay, the mutated residues do not appear to be functionally necessary. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonistic properties, supporting our hypothesis that the channel between helices 1 and 3 is involved in a second receptor binding site. We then analyzed the antagonistic and self-antagonistic properties of native hPRL and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (approximately 10 microM), no self-inhibition was observed with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL receptor interaction which slightly differs from that proposed earlier for the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (1992) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two binding sites of hGH, about the same binding affinity, thus preventing self-antagonism at high concentrations. PMID:7798264

  1. Novel mechanisms of intracellular cholesterol transport: oxysterol-binding proteins and membrane contact sites.

    PubMed

    Du, Ximing; Brown, Andrew J; Yang, Hongyuan

    2015-08-01

    Cholesterol is an essential membrane constituent, and also plays a key role in cell signalling. Within a cell, how cholesterol is transported and how its heterogeneous distribution is maintained are poorly understood. Recent advances have identified novel pathways and regulators of cholesterol trafficking. Sterol transfer by lipid-binding proteins, such as OSBP (oxysterol-binding protein), coupled with phosphatidylinositol 4-phosphate exchange at membrane contact sites (MCSs) has emerged as a new theme of cholesterol transport between organellar membranes. Moreover, a previously unappreciated role of peroxisomes in cholesterol trafficking has been revealed recently. These discoveries highlight the crucial role of MCSs, or junctions, in facilitating lipid movement, and provide mechanistic insights into how cholesterol is sorted in cells. PMID:25932595

  2. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

    PubMed Central

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  3. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    PubMed

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

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

    PubMed Central

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

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  5. Autoradiographic localization of relaxin binding sites in rat brain

    SciTech Connect

    Osheroff, P.L.; Phillips, H.S. )

    1991-08-01

    Relaxin is a member of the insulin family of polypeptide hormones and exerts its best understood actions in the mammalian reproductive system. Using a biologically active 32P-labeled human relaxin, the authors have previously shown by in vitro autoradiography specific relaxin binding sites in rat uterus, cervix, and brain tissues. Using the same approach, they describe here a detailed localization of human relaxin binding sites in the rat brain. Displaceable relaxin binding sites are distributed in discrete regions of the olfactory system, neocortex, hypothalamus, hippocampus, thalamus, amygdala, midbrain, and medulla of the male and female rat brain. Characterization of the relaxin binding sites in the subfornical organ and neocortex reveals a single class of high-affinity sites (Kd = 1.4 nM) in both regions. The binding of relaxin to two of the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminalis) and the neurosecretory magnocellular hypothalamic nuclei (i.e., paraventricular and supraoptic nuclei) provides the anatomical and biochemical basis for emerging physiological evidence suggesting a central role for relaxin in the control of blood pressure and hormone release. They conclude that specific, high-affinity relaxin binding sites are present in discrete regions of the rat brain and that the distribution of some of these sites may be consistent with a role for relaxin in control of vascular volume and blood pressure.

  6. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; dos Santos, Daniel J V A

    2013-07-22

    P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data. PMID:23802684

  7. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    PubMed Central

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-01

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor’s oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  8. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI.

    PubMed

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-14

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  9. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    SciTech Connect

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

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

    SciTech Connect

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. )

    1991-07-01

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

  11. Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

    PubMed Central

    Heyse, S.; Vogel, H.; Sänger, M.; Sigrist, H.

    1995-01-01

    A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde

  12. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    PubMed

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes. PMID:25966259

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

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

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

  14. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding.

    PubMed

    Liko, Idlir; Degiacomi, Matteo T; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V

    2016-07-19

    Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance. PMID:27364008

  15. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

    PubMed Central

    Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

    2016-01-01

    Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance. PMID:27364008

  16. GPS-Lipid: a robust tool for the prediction of multiple lipid modification sites.

    PubMed

    Xie, Yubin; Zheng, Yueyuan; Li, Hongyu; Luo, Xiaotong; He, Zhihao; Cao, Shuo; Shi, Yi; Zhao, Qi; Xue, Yu; Zuo, Zhixiang; Ren, Jian

    2016-01-01

    As one of the most common post-translational modifications in eukaryotic cells, lipid modification is an important mechanism for the regulation of variety aspects of protein function. Over the last decades, three classes of lipid modifications have been increasingly studied. The co-regulation of these different lipid modifications is beginning to be noticed. However, due to the lack of integrated bioinformatics resources, the studies of co-regulatory mechanisms are still very limited. In this work, we developed a tool called GPS-Lipid for the prediction of four classes of lipid modifications by integrating the Particle Swarm Optimization with an aging leader and challengers (ALC-PSO) algorithm. GPS-Lipid was proven to be evidently superior to other similar tools. To facilitate the research of lipid modification, we hosted a publicly available web server at http://lipid.biocuckoo.org with not only the implementation of GPS-Lipid, but also an integrative database and visualization tool. We performed a systematic analysis of the co-regulatory mechanism between different lipid modifications with GPS-Lipid. The results demonstrated that the proximal dual-lipid modifications among palmitoylation, myristoylation and prenylation are key mechanism for regulating various protein functions. In conclusion, GPS-lipid is expected to serve as useful resource for the research on lipid modifications, especially on their co-regulation. PMID:27306108

  17. GPS-Lipid: a robust tool for the prediction of multiple lipid modification sites

    PubMed Central

    Xie, Yubin; Zheng, Yueyuan; Li, Hongyu; Luo, Xiaotong; He, Zhihao; Cao, Shuo; Shi, Yi; Zhao, Qi; Xue, Yu; Zuo, Zhixiang; Ren, Jian

    2016-01-01

    As one of the most common post-translational modifications in eukaryotic cells, lipid modification is an important mechanism for the regulation of variety aspects of protein function. Over the last decades, three classes of lipid modifications have been increasingly studied. The co-regulation of these different lipid modifications is beginning to be noticed. However, due to the lack of integrated bioinformatics resources, the studies of co-regulatory mechanisms are still very limited. In this work, we developed a tool called GPS-Lipid for the prediction of four classes of lipid modifications by integrating the Particle Swarm Optimization with an aging leader and challengers (ALC-PSO) algorithm. GPS-Lipid was proven to be evidently superior to other similar tools. To facilitate the research of lipid modification, we hosted a publicly available web server at http://lipid.biocuckoo.org with not only the implementation of GPS-Lipid, but also an integrative database and visualization tool. We performed a systematic analysis of the co-regulatory mechanism between different lipid modifications with GPS-Lipid. The results demonstrated that the proximal dual-lipid modifications among palmitoylation, myristoylation and prenylation are key mechanism for regulating various protein functions. In conclusion, GPS-lipid is expected to serve as useful resource for the research on lipid modifications, especially on their co-regulation. PMID:27306108

  18. Binding sites for gonadotropins in human postmenopausal ovaries

    SciTech Connect

    Nakano, R.; Shima, K.; Yamoto, M.; Kobayashi, M.; Nishimori, K.; Hiraoka, J.

    1989-02-01

    The binding of human LH and human FSH to postmenopausal ovarian tissue from 21 patients with cervical carcinoma was analyzed. The binding sites for FSH and LH were demonstrated in postmenopausal ovarian tissue. The surface-binding sites for gonadotropins were localized in the cells of cortical stroma of the postmenopausal ovary. In addition, diffuse cytoplasmic staining of endogenous estrogen and 3 beta-hydroxysteroid dehydrogenase activity were detected immunohistochemically and histochemically in the cells of the cortical stroma. Electron microscopic study also suggested steroidogenic function in the cells of the cortical stroma. The results of the present study suggest that postmenopausal ovaries contain specific binding sites for pituitary gonadotropins and play a role in ovarian steroidogenesis.

  19. Partial characterization of specific cantharidin binding sites in mouse tissues

    SciTech Connect

    Graziano, M.J.; Pessah, I.N.; Matsuzawa, M.; Casida, J.E.

    1988-06-01

    The mode of action of cantharidin, the natural vesicant of blister beetles, is examined by radioligand binding studies with mouse tissues. (3H)Cantharidin undergoes specific and saturable binding with the liver cytosol, which is characterized as follows: Kd and Bmax values of 30 nM and 1.8 pmol/mg of protein, respectively; linearity with respect to protein concentration; pH optimum of 6.5 to 7.5; association and dissociation half-times of 20 min and 12 hr, respectively; and 50% inhibition by Mg2+ at 70 microM, Ca2+ at 224 microM, pyrophosphate at 27 microM, and nucleotide triphosphates at 52-81 microM. The binding site undergoes a loss of activity at 45 degrees or higher. The toxicological relevance of this specific (3H)cantharidin binding site of mouse liver cytosol is established in three ways. First, the potency of 15 active cantharidin analogs for inhibiting (3H)cantharidin binding is correlated with their acute toxicity to mice (r = 0.829). Second, 26 related compounds that are inactive in inhibiting (3H)cantharidin binding are also of little or no toxicity to mice. Finally, the binding of (3H) cantharidin to liver cytosol from mice poisoned with increasing amounts of unlabeled cantharidin is inhibited in a dose-dependent manner. (3H)Cantharidin also specifically binds to cytosol fractions of blood, brain, heart, kidney, lung, pancreas, skin, spleen, and stomach. The characteristics of the specific binding site in brain are very similar to those determined in liver with respect to Kd, Bmax, association/dissociation kinetics, and sensitivity to inhibitors. It therefore appears that the toxicity of cantharidin and related oxabicycloheptanes, including the herbicide endothal, is attributable to binding at a specific site in liver and possibly other tissues.

  20. MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models.

    PubMed

    Nastou, Katerina C; Tsaousis, Georgios N; Papandreou, Nikos C; Hamodrakas, Stavros J

    2016-07-01

    A large number of modular domains that exhibit specific lipid binding properties are present in many membrane proteins involved in trafficking and signal transduction. These domains are present in either eukaryotic peripheral membrane or transmembrane proteins and are responsible for the non-covalent interactions of these proteins with membrane lipids. Here we report a profile Hidden Markov Model based method capable of detecting Membrane Binding Proteins (MBPs) from information encoded in their amino acid sequence, called MBPpred. The method identifies MBPs that contain one or more of the Membrane Binding Domains (MBDs) that have been described to date, and further classifies these proteins based on their position in respect to the membrane, either as peripheral or transmembrane. MBPpred is available online at http://bioinformatics.biol.uoa.gr/MBPpred. This method was applied in selected eukaryotic proteomes, in order to examine the characteristics they exhibit in various eukaryotic kingdoms and phyla. PMID:27048983

  1. Functional conservation of Rel binding sites in drosophilid genomes

    PubMed Central

    Copley, Richard R.; Totrov, Maxim; Linnell, Jane; Field, Simon; Ragoussis, Jiannis; Udalova, Irina A.

    2007-01-01

    Evolutionary constraints on gene regulatory elements are poorly understood: Little is known about how the strength of transcription factor binding correlates with DNA sequence conservation, and whether transcription factor binding sites can evolve rapidly while retaining their function. Here we use the model of the NFKB/Rel-dependent gene regulation in divergent Drosophila species to examine the hypothesis that the functional properties of authentic transcription factor binding sites are under stronger evolutionary constraints than the genomic background. Using molecular modeling we compare tertiary structures of the Drosophila Rel family proteins Dorsal, Dif, and Relish and demonstrate that their DNA-binding and protein dimerization domains undergo distinct rates of evolution. The accumulated amino acid changes, however, are unlikely to affect DNA sequence recognition and affinity. We employ our recently developed microarray-based experimental platform and principal coordinates statistical analysis to quantitatively and systematically profile DNA binding affinities of three Drosophila Rel proteins to 10,368 variants of the NFKB recognition sequences. We then correlate the evolutionary divergence of gene regulatory regions with differences in DNA binding affinities. Genome-wide analyses reveal a significant increase in the number of conserved Rel binding sites in promoters of developmental and immune genes. Significantly, the affinity of Rel proteins to these sites was higher than to less conserved sites and was maintained by the conservation of the DNA binding site sequence (static conservation) or in some cases despite significantly diverged sequences (dynamic conservation). We discuss how two types of conservation may contribute to the stabilization and optimization of a functional gene regulatory code in evolution. PMID:17785540

  2. Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

    PubMed Central

    Quon, Evan; Beh, Christopher T.

    2015-01-01

    Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

  3. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design. PMID:27399277

  4. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites

    SciTech Connect

    Lesch, K.P.; Schott, W.; Gross, S.

    1987-09-01

    The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using /sup 125/I-labeled estradiol (/sup 125/I-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for /sup 125/I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis.

  5. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    PubMed

    Kovalevskaya, Nadezda V; Bokhovchuk, Fedir M; Vuister, Geerten W

    2012-06-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845-2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca(2+)-concentrations. PMID:22354706

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

    PubMed Central

    Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H.

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/. PMID:26987123

  7. 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

  8. 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

  9. Sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition

    SciTech Connect

    Feller, D.J.; Talvenheimo, J.A.; Catterall, W.A.

    1985-09-25

    Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated SSNa influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Binding of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which (Na+)out/(Na+)in is varied by changing (Na+)in or (Na+)out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.

  10. Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

    PubMed

    Gupta, Shikhar; Fallarero, Adyary; Järvinen, Päivi; Karlsson, Daniela; Johnson, Mark S; Vuorela, Pia M; Mohan, C Gopi

    2011-02-15

    Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 μM) and Spec2 (IC(50)=5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity. PMID:21273074

  11. Identification and characterization of anion binding sites in RNA.

    PubMed

    Kieft, Jeffrey S; Chase, Elaine; Costantino, David A; Golden, Barbara L

    2010-06-01

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions. PMID:20410239

  12. Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes.

    PubMed

    Biswas, Nupur; Bhattacharya, Rupak; Saha, Arindam; Jana, Nikhil R; Basu, Jaydeep K

    2015-10-01

    Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes. PMID:26327393

  13. The Effect of Environment on the Recognition and Binding of Vancomycin to Native and Resistant Forms of Lipid II

    PubMed Central

    Jia, ZhiGuang; O'Mara, Megan L.; Zuegg, Johannes; Cooper, Matthew A.; Mark, Alan E.

    2011-01-01

    Molecular dynamics simulations and free energy calculations have been used to examine in detail the mechanism by which a receptor molecule (the glycopeptide antibiotic vancomycin) recognizes and binds to a target molecule (lipid II) embedded within a membrane environment. The simulations show that the direct interaction of vancomycin with lipid II, as opposed to initial binding to the membrane, leads most readily to the formation of a stable complex. The recognition of lipid II by vancomycin occurred via the N-terminal amine group of vancomycin and the C-terminal carboxyl group of lipid II. Despite lying at the membrane-water interface, the interaction of vancomycin with lipid II was found to be essentially identical to that of soluble tripeptide analogs of lipid II (Ac-d-Ala-d-Ala; root mean-square deviation 0.11 nm). Free energy calculations also suggest that the relative binding affinity of vancomycin for native, resistant, and synthetic forms of membrane-bound lipid II was unaffected by the membrane environment. The effect of the dimerization of vancomycin on the binding of lipid II, the position of lipid II within a biological membrane, and the effect of the isoamylene tail of lipid II on membrane fluidity have also been examined. PMID:22261057

  14. Acetylation within the First 17 Residues of Huntingtin Exon 1 Alters Aggregation and Lipid Binding.

    PubMed

    Chaibva, Maxmore; Jawahery, Sudi; Pilkington, Albert W; Arndt, James R; Sarver, Olivia; Valentine, Stephen; Matysiak, Silvina; Legleiter, Justin

    2016-07-26

    Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization. As there are three lysines within Nt(17), we evaluated the impact of lysine acetylation on htt aggregation in solution and on model lipid bilayers. Acetylation of htt-exon1(51Q) and synthetic truncated htt-exon 1 mimicking peptides (Nt(17)-Q35-P10-KK) was achieved using a selective covalent label, sulfo-N-hydroxysuccinimide (NHSA). With this treatment, all three lysine residues (K6, K9, and K15) in Nt(17) were significantly acetylated. N-terminal htt acetylation retarded fibril formation in solution and promoted the formation of larger globular aggregates. Acetylated htt also bound lipid membranes and disrupted the lipid bilayer morphology less aggressively compared with the wild-type. Computational studies provided mechanistic insights into how acetylation alters the interaction of Nt(17) with lipid membranes. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers. PMID:27463137

  15. Penicillin-binding site on the Escherichia coli cell envelope

    SciTech Connect

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-08-01

    The binding of /sup 35/S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin.

  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. From fatty-acid sensing to chylomicron synthesis: role of intestinal lipid-binding proteins.

    PubMed

    Buttet, Marjorie; Traynard, Véronique; Tran, Thi Thu Trang; Besnard, Philippe; Poirier, Hélène; Niot, Isabelle

    2014-01-01

    Today, it is well established that the development of obesity and associated diseases results, in part, from excessive lipid intake associated with a qualitative imbalance. Among the organs involved in lipid homeostasis, the small intestine is the least studied even though it determines lipid bioavailability and largely contributes to the regulation of postprandial hyperlipemia (triacylglycerols (TG) and free fatty acids (FFA)). Several Lipid-Binding Proteins (LBP) are expressed in the small intestine. Their supposed intestinal functions were initially based on what was reported in other tissues, and took no account of the physiological specificity of the small intestine. Progressively, the identification of regulating factors of intestinal LBP and the description of the phenotype of their deletion have provided new insights into cellular and molecular mechanisms involved in fat absorption. This review will discuss the physiological contribution of each LBP in the main steps of intestinal absorption of long-chain fatty acids (LCFA): uptake, trafficking and reassembly into chylomicrons (CM). Moreover, current data indicate that the small intestine is able to adapt its lipid absorption capacity to the fat content of the diet, especially through the coordinated induction of LBP. This adaptation requires the existence of a mechanism of intestinal lipid sensing. Emerging data suggest that the membrane LBP CD36 may operate as a lipid receptor that triggers an intracellular signal leading to the modulation of the expression of LBP involved in CM formation. This event could be the starting point for the optimized synthesis of large CM, which are efficiently degraded in blood. Better understanding of this intestinal lipid sensing might provide new approaches to decrease the prevalence of postprandial hypertriglyceridemia, which is associated with cardiovascular diseases, insulin resistance and obesity. PMID:23958439

  18. Allosteric interaction of trimebutine maleate with dihydropyridine binding sites.

    PubMed

    Nagasaki, M; Kurosawa, H; Naito, K; Tamaki, H

    1990-07-31

    The effects of trimebutine maleate on [3H]nitrendipine binding to guinea-pig ileal smooth muscle membranes and Ca2(+)-induced contraction of the taenia cecum were studied. Specific binding of [3H]nitrendipine to smooth muscle membranes was saturable, with a KD value and maximum number of binding sites (Bmax) of 0.16 nM and 1070 fmol/mg protein, respectively. Trimebutine inhibited [3H]nitrendipine binding in a concentration-dependent manner with a Ki value of 9.3 microM. In the presence of trimebutine (10 microM), Scatchard analysis indicated a competitive-like inhibition with a decrease in the binding affinity (0.31 nM) without a change in Bmax (1059 fmol/mg protein). However, a dissociation experiment using trimebutine (10 or 100 microM) showed that the decreased affinity was due to an increase of the dissociation rate constant of [3H]nitrendipine binding to the membrane. In mechanical experiments using the taenia cecum, trimebutine (3-30 microM) caused a parallel rightward shift of the dose-response curve for the contractile response to a higher concentration range of Ca2+ under high-K+ conditions in a noncompetitive manner. These results suggest that trimebutine has negative allosteric interactions with 1,4-dihydropyridine binding sites on voltage-dependent Ca2+ channels and antagonizes Ca2+ influx, consequently inhibiting contractions of intestinal smooth muscle. PMID:2171963

  19. Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

    SciTech Connect

    Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.; Janis, R.A. State Univ. of New York, Buffalo )

    1991-03-11

    The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{sup 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.

  20. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  1. Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature

    NASA Astrophysics Data System (ADS)

    Junesch, Juliane; Emilsson, Gustav; Xiong, Kunli; Kumar, Shailabh; Sannomiya, Takumi; Pace, Hudson; Vörös, Janos; Oh, Sang-Hyun; Bally, Marta; Dahlin, Andreas B.

    2015-09-01

    The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic ``nanowells'' provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic ``nanowells'' provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids. Electronic supplementary information (ESI) available: Additional plasmonic sensing results, numerical electromagnetic simulations, quartz crystal microbalance data, fluorescence recovery after photobleaching, additional electron microscopy images, experimental methodology and materials used. See DOI: 10.1039/c5nr04208a

  2. A sterol binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing

    PubMed Central

    Mousley, Carl J.; Yuan, Peihua; Gaur, Naseem A.; Trettin, Kyle D.; Nile, Aaron H.; Deminoff, Stephen J.; Dewar, Brian J.; Wolpert, Max; Macdonald, Jeffrey M.; Herman, Paul K.; Hinnebusch, Alan G.; Bankaitis, Vytas A.

    2012-01-01

    SUMMARY Kes1, and other oxysterol binding protein (OSBP) superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids, and dampens gene expression driven by Gcn4; the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic ‘large Mediator’ complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing. PMID:22341443

  3. Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G

    PubMed Central

    Im, Jae Hong; Nakane, Takashi; Yanagishita, Hiroshi; Ikegami, Toru; Kitamoto, Dai

    2001-01-01

    Background There have been many attempts to develop new materials with stability and high affinity towards immunoglobulins. Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins. However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts. We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A) and human immunoglobulin G (HIgG). Results In ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1. Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A. The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate) (polyHEMA) beads, whereas the amount of human serum albumin slightly decreased. Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution. The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 × 106 (M-1) for HIgG, which is approximately 4-fold greater than that of protein A reported. Conclusions MEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio. This is the first report on the binding of a natural human antibody towards a yeast glycolipid. PMID:11604104

  4. Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels.

    PubMed Central

    Rokitskaya, T I; Block, M; Antonenko, Y N; Kotova, E A; Pohl, P

    2000-01-01

    The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS(2) was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AlPcS(1), AlPcS(2), AlPcS(3), and AlPcS(4), respectively. At high concentrations alpha was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcS(n) (2 binding to LUVs and sensitized photoinactivation of gramicidin channels. It is concluded that photosensitizer binding to membrane lipids is a prerequisite for the photodynamic inactivation of gramicidin channels. PMID:10777753

  5. Identification of the endothelial cell binding site for factor IX.

    PubMed Central

    Cheung, W F; van den Born, J; Kühn, K; Kjellén, L; Hudson, B G; Stafford, D W

    1996-01-01

    We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined. PMID:8855310

  6. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

    PubMed Central

    Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

    2015-01-01

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

  7. Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.

    PubMed

    Pérez Santero, Silvia; Favretto, Filippo; Zanzoni, Serena; Chignola, Roberto; Assfalg, Michael; D'Onofrio, Mariapina

    2016-09-15

    Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell. PMID:27457417

  8. The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix

    PubMed Central

    Barneda, David; Planas-Iglesias, Joan; Gaspar, Maria L; Mohammadyani, Dariush; Prasannan, Sunil; Dormann, Dirk; Han, Gil-Soo; Jesch, Stephen A; Carman, George M; Kagan, Valerian; Parker, Malcolm G; Ktistakis, Nicholas T; Klein-Seetharaman, Judith; Dixon, Ann M; Henry, Susan A; Christian, Mark

    2015-01-01

    Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat. DOI: http://dx.doi.org/10.7554/eLife.07485.001 PMID:26609809

  9. The RNA binding site of bacteriophage MS2 coat protein.

    PubMed Central

    Peabody, D S

    1993-01-01

    The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat. Images PMID:8440248

  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. Specific binding sites for muramyl peptides on murine macrophages

    SciTech Connect

    Silverman, D.H.S.; Krueger, J.M.; Karnovsky, M.L.

    1986-03-15

    Two radiolabeled (/sup 125/I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and K/sub D/ values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

  12. Characterization of Heparin-binding Site of Tissue Transglutaminase

    PubMed Central

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

    2012-01-01

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

  13. Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

    PubMed Central

    Nayak, Tapan K.; Bruhova, Iva; Chakraborty, Srirupa; Gupta, Shaweta; Zheng, Wenjun; Auerbach, Anthony

    2014-01-01

    A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is ∼−2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse. PMID:25422413

  14. The binding versatility of plant acyl-CoA-binding proteins and their significance in lipid metabolism.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-09-01

    Acyl-CoA esters are the activated form of fatty acids and play important roles in lipid metabolism and the regulation of cell functions. They are bound and transported by nonenzymic proteins such as the acyl-CoA-binding proteins (ACBPs). Although plant ACBPs were so named by virtue of amino acid homology to existing yeast and mammalian counterparts, recent studies revealed that ligand specificities of plant ACBPs are not restricted to acyl-CoA esters. Arabidopsis and rice ACBPs also interact with phospholipids, and their affinities to different acyl-CoA species and phospholipid classes vary amongst isoforms. Their ligands also include heavy metals. Interactors of plant ACBPs are further diversified due to the evolution of protein-protein interacting domains. This review summarizes our current understanding of plant ACBPs with a focus on their binding versatility. Their broad ligand range is of paramount significance in serving a multitude of functions during development and stress responses as discussed herein. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26747650

  15. Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array.

    PubMed

    Worstell, Nolan C; Krishnan, Pratik; Weatherston, Joshua D; Wu, Hung-Jen

    2016-01-01

    Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms. PMID:27070150

  16. Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

    PubMed Central

    Weatherston, Joshua D.

    2016-01-01

    Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms. PMID:27070150

  17. Thymocyte plasma membrane: the location of specific glucocorticoid binding sites

    SciTech Connect

    Sergeev, P.V.; Kalinin, G.V.; Dukhanin, A.S.

    1987-01-01

    In modern molecular endocrinology it is now possible to determine the localization of receptors for biologically active substances with the aid of ligands, with high affinity for the receptor, immobilized on polymers. The purpose of this paper is to study the ability of hydrocortisone (HC), immobilized on polyvinylpyrrolidone (PVP-HC), to reduce binding of tritium-HC by thymocytes of adrenalectomized rats. It is determined that specific binding sites for HC on rat thymocytes are also accessible for PVP-HC, which, due to the fact that this immobilized version of HC does not penetrate into the cell, leads to the conclusion that the binding sites for HC itself are located in the plasma membrane.

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

    PubMed Central

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements. PMID:21087992

  19. Reliable prediction of transcription factor binding sites by phylogenetic verification.

    PubMed

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H

    2005-11-22

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is capable of accurately detecting TF binding sites, it also enhances our ability to predict the cis-regulatory modules. We applied this method on the published chromatin immunoprecipitation (ChIP)-chip data in Saccharomyces cerevisiae and found that its sensitivity and specificity are 9% and 14% higher than those of two recent methods. We also recovered almost all of the previously verified TF binding sites and made predictions on the cis-regulatory elements that govern the tight regulation of ribosomal protein genes in 13 eukaryote species (2 plants, 4 yeasts, 2 worms, 2 insects, and 3 mammals). These results give insights to the transcriptional regulation in eukaryotic organisms. PMID:16286651

  20. Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2003-11-11

    Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. PMID:14596623

  1. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    PubMed Central

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  2. Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations.

    PubMed

    Hedger, George; Sansom, Mark S P

    2016-10-01

    Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterization of these sites are of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946244

  3. Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties.

    PubMed

    Schwieger, Christian; Achilles, Anja; Scholz, Sven; Rüger, Jan; Bacia, Kirsten; Saalwaechter, Kay; Kressler, Jörg; Blume, Alfred

    2014-09-01

    A novel class of symmetric amphi- and triphilic (hydrophilic, lipophilic, fluorophilic) block copolymers has been investigated with respect to their interactions with lipid membranes. The amphiphilic triblock copolymer has the structure PGMA(20)-PPO(34)-PGMA(20) (GP) and it becomes triphilic after attaching perfluoroalkyl moieties (F9) to either end which leads to F(9)-PGMA(20)-PPO(34)-PGMA(20)-F(9) (F-GP). The hydrophobic poly(propylene oxide) (PPO) block is sufficiently long to span a lipid bilayer. The poly(glycerol monomethacrylate) (PGMA) blocks have a high propensity for hydrogen bonding. The hydrophobic and lipophobic perfluoroalkyl moieties have the tendency to phase segregate in aqueous as well as in hydrocarbon environments. We performed differential scanning calorimetry (DSC) measurements on polymer bound lipid vesicles under systematic variation of the bilayer thickness, the nature of the lipid headgroup, and the polymer concentration. The vesicles were composed of phosphatidylcholines (DMPC, DPPC, DAPC, DSPC) or phosphatidylethanolamines (DMPE, DPPE, POPE). We showed that GP as well as F-GP binding have membrane stabilizing and destabilizing components. PPO and F9 blocks insert into the hydrophobic part of the membrane concomitantly with PGMA block adsorption to the lipid headgroup layer. The F9 chains act as additional membrane anchors. The insertion of the PPO blocks of both GP and F-GP could be proven by 2D-NOESY NMR spectroscopy. By fluorescence microscopy we show that F-GP binding increases the porosity of POPC giant unilamellar vesicles (GUVs), allowing the influx of water soluble dyes as well as the translocation of the complete triphilic polymer and its accumulation at the GUV surface. These results open a new route for the rational design of membrane systems with specific properties. PMID:24942348

  4. Cholesterol Modifies Huntingtin Binding to, Disruption of, and Aggregation on Lipid Membranes.

    PubMed

    Gao, Xiang; Campbell, Warren A; Chaibva, Maxmore; Jain, Pranav; Leslie, Ashley E; Frey, Shelli L; Legleiter, Justin

    2016-01-12

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ domains are directly correlated to disease-related htt aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes that are predominantly comprised of lipids. Since cholesterol homeostasis is altered in HD, we investigated how varying cholesterol content modifies the interactions between htt and lipid membranes. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added cholesterol. As the cholesterol content of the membrane increased, the extent of htt insertion decreased. Vesicles containing extra cholesterol were resistant to htt-induced permeabilization. Morphological and mechanical changes in the bilayer associated with exposure to htt were also drastically altered by the presence of cholesterol. Disrupted regions of pure TBLE bilayers were grainy in appearance and associated with a large number of globular aggregates. In contrast, morphological changes induced by htt in bilayers enriched in cholesterol were plateau-like with a smooth appearance. Collectively, these observations suggest that the presence and amount of cholesterol in lipid membranes play a critical role in htt binding and aggregation on lipid membranes. PMID:26652744

  5. The human "magnesome": detecting magnesium binding sites on human proteins

    PubMed Central

    2012-01-01

    Background Magnesium research is increasing in molecular medicine due to the relevance of this ion in several important biological processes and associated molecular pathogeneses. It is still difficult to predict from the protein covalent structure whether a human chain is or not involved in magnesium binding. This is mainly due to little information on the structural characteristics of magnesium binding sites in proteins and protein complexes. Magnesium binding features, differently from those of other divalent cations such as calcium and zinc, are elusive. Here we address a question that is relevant in protein annotation: how many human proteins can bind Mg2+? Our analysis is performed taking advantage of the recently implemented Bologna Annotation Resource (BAR-PLUS), a non hierarchical clustering method that relies on the pair wise sequence comparison of about 14 millions proteins from over 300.000 species and their grouping into clusters where annotation can safely be inherited after statistical validation. Results After cluster assignment of the latest version of the human proteome, the total number of human proteins for which we can assign putative Mg binding sites is 3,751. Among these proteins, 2,688 inherit annotation directly from human templates and 1,063 inherit annotation from templates of other organisms. Protein structures are highly conserved inside a given cluster. Transfer of structural properties is possible after alignment of a given sequence with the protein structures that characterise a given cluster as obtained with a Hidden Markov Model (HMM) based procedure. Interestingly a set of 370 human sequences inherit Mg2+ binding sites from templates sharing less than 30% sequence identity with the template. Conclusion We describe and deliver the "human magnesome", a set of proteins of the human proteome that inherit putative binding of magnesium ions. With our BAR-hMG, 251 clusters including 1,341 magnesium binding protein structures

  6. Variable structure motifs for transcription factor binding sites

    PubMed Central

    2010-01-01

    Background Classically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. Results We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. Conclusions We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable

  7. Targeting Different Transthyretin Binding Sites with Unusual Natural Compounds.

    PubMed

    Ortore, Gabriella; Orlandini, Elisabetta; Braca, Alessandra; Ciccone, Lidia; Rossello, Armando; Martinelli, Adriano; Nencetti, Susanna

    2016-08-19

    Misfolding and aggregation of the transthyretin (TTR) protein leads to certain forms of amyloidosis. Some nutraceuticals, such as flavonoids and natural polyphenols, have recently been investigated as modulators of the self-assembly process of TTR, but they generally suffer from limited bioavailability. To discover innovative and more bioavailable natural compounds able to inhibit TTR amyloid formation, a docking study was performed using the crystallographic structure of TTR. This computational strategy was projected as an ad hoc inspection of the possible relationship between binding site location and modulation of the assembly process; interactions with the as-yet-unexplored epigallocatechin gallate (EGCG) sites and with the thyroxine (T4) pocket were simultaneously analyzed. All the compounds studied seem to prefer the traditional T4 binding site, but some interesting results emerged from the screening of an in-house database, used for validating the computational protocol, and of the Herbal Ingredients Targets (HIT) catalogue available on the ZINC database. PMID:27159149

  8. 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

  9. Unquenchable Surface Potential Dramatically Enhances Cu(2+) Binding to Phosphatidylserine Lipids.

    PubMed

    Cong, Xiao; Poyton, Matthew F; Baxter, Alexis J; Pullanchery, Saranya; Cremer, Paul S

    2015-06-24

    Herein, the apparent equilibrium dissociation constant, K(Dapp), between Cu(2+) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), a negatively charged phospholipid, was measured as a function of PS concentrations in supported lipid bilayers (SLBs). The results indicated that K(Dapp) for Cu(2+) binding to PS-containing SLBs was enhanced by a factor of 17,000 from 110 nM to 6.4 pM as the PS density in the membrane was increased from 1.0 to 20 mol %. Although Cu(2+) bound bivalently to POPS at higher PS concentrations, this was not the dominant factor in increasing the binding affinity. Rather, the higher concentration of Cu(2+) within the double layer above the membrane was largely responsible for the tightening. Unlike the binding of other divalent metal ions such as Ca(2+) and Mg(2+) to PS, Cu(2+) binding does not alter the net negative charge on the membrane as the Cu(PS)2 complex forms. As such, the Cu(2+) concentration within the double layer region was greatly amplified relative to its concentration in bulk solution as the PS density was increased. This created a far larger enhancement to the apparent binding affinity than is observed by standard multivalent effects. These findings should help provide an understanding on the extent of Cu(2+)-PS binding in cell membranes, which may be relevant to biological processes such as amyloid-β peptide toxicity and lipid oxidation. PMID:26065920

  10. 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.

  11. 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

  12. 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

  13. Binding site of MraZ transcription factor in Mollicutes.

    PubMed

    Fisunov, G Y; Evsyutina, D V; Semashko, T A; Arzamasov, A A; Manuvera, V A; Letarov, A V; Govorun, V M

    2016-06-01

    Mollicutes (mycoplasmas) feature a significant loss of known regulators of gene expression. Here, we identified the recognition site of the MraZ-family regulator of Mycoplasma gallisepticum, which is conserved in many species of different clades within class Mollicutes. The MraZ binding site is AAAGTG[T/G], in the promoter of mraZ gene it forms a series of direct repeats with a structure (AAAGTG[T/G]N3)k, where k = 3 most frequently. MraZ binds to a single repeat as an octamer complex. MraZ can also bind a single binding site or a series of repeats with different spacer lengths (2-4 nt); thus, it may play a role in the regulation of multiple operons in Mollicutes. In M. gallisepticum, MraZ acts as a transcriptional activator. The overexpression of MraZ leads to moderate filamentation of cells and the formation of aggregates, likely as a result of incomplete cytokinesis. PMID:26945841

  14. Ion Binding Sites and their Representations by Reduced Models

    PubMed Central

    Roux, Benoît

    2013-01-01

    The binding of small metal ions to complex macromolecular structures is typically dominated by strong local interactions of the ion with its nearest ligands. Progress in understanding the molecular determinants of ion selectivity can often be achieved by considering simplified reduced models comprised of only the most important ion-coordinating ligands. Although the main ingredients underlying simplified reduced models are intuitively clear, a formal statistical mechanical treatment is nonetheless necessary in order to draw meaningful conclusions about complex macromolecular systems. By construction, reduced models only treat the ion and the nearest coordinating ligands explicitly. The influence of the missing atoms from the protein or the solvent is incorporated indirectly. Quasi-chemical theory offers one example of how to carry out such a separation in the case of ion solvation in bulk liquids, and in several ways, a statistical mechanical formulation of reduced binding site models for macromolecules is expected to follow a similar route. However, there are also important differences when the ion-coordinating moieties are not solvent molecules from a bulk phase, but are molecular ligands covalently bonded to a macromolecular structure. Here, a statistical mechanical formulation of reduced binding site models is elaborated to address these issues. The formulation provides a useful framework to construct reduced binding site models, and define the average effect from the surroundings on the ion and the nearest coordinating ligands. PMID:22494321

  15. Computational investigation of cholesterol binding sites on mitochondrial VDAC.

    PubMed

    Weiser, Brian P; Salari, Reza; Eckenhoff, Roderic G; Brannigan, Grace

    2014-08-21

    The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs. We docked cholesterol to specific sites on VDAC that were previously identified with NMR, and we tested the reliability of multiple docking results in each site with simulations. The most favorable binding modes were used to build a VDAC model with cholesterol occupying five unique sites, and during multiple 100 ns simulations, cholesterol stably and reproducibly remained bound to the protein. For comparison, VDAC was simulated in systems with identical components but with cholesterol initially unbound. The dynamics of loops that connect adjacent β-strands were most affected by bound cholesterol, with the averaged root-mean-square fluctuation (RMSF) of multiple residues altered by 20-30%. Cholesterol binding also stabilized charged residues inside the channel and localized the surrounding electrostatic potentials. Despite this, ion diffusion through the channel was not significantly affected by bound cholesterol, as evidenced by multi-ion potential of mean force measurements. Although we observed modest effects of cholesterol on the open channel, our model will be particularly useful in experiments that investigate how cholesterol affects VDAC function under applied electrochemical forces and also how other ligands and proteins interact with the channel. PMID:25080204

  16. Detection of Binding Site Molecular Interaction Field Similarities.

    PubMed

    Chartier, Matthieu; Najmanovich, Rafael

    2015-08-24

    Protein binding-site similarity detection methods can be used to predict protein function and understand molecular recognition, as a tool in drug design for drug repurposing and polypharmacology, and for the prediction of the molecular determinants of drug toxicity. Here, we present IsoMIF, a method able to identify binding site molecular interaction field similarities across protein families. IsoMIF utilizes six chemical probes and the detection of subgraph isomorphisms to identify geometrically and chemically equivalent sections of protein cavity pairs. The method is validated using six distinct data sets, four of those previously used in the validation of other methods. The mean area under the receiver operator curve (AUC) obtained across data sets for IsoMIF is higher than those of other methods. Furthermore, while IsoMIF obtains consistently high AUC values across data sets, other methods perform more erratically across data sets. IsoMIF can be used to predict function from structure, to detect potential cross-reactivity or polypharmacology targets, and to help suggest bioisosteric replacements to known binding molecules. Given that IsoMIF detects spatial patterns of molecular interaction field similarities, its predictions are directly related to pharmacophores and may be readily translated into modeling decisions in structure-based drug design. IsoMIF may in principle detect similar binding sites with distinct amino acid arrangements that lead to equivalent interactions within the cavity. The source code to calculate and visualize MIFs and MIF similarities are freely available. PMID:26158641

  17. Photoaffinity labeling in target- and binding-site identification

    PubMed Central

    Smith, Ewan; Collins, Ian

    2015-01-01

    Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

  18. De-Novo Identification of PPARγ/RXR Binding Sites and Direct Targets during Adipogenesis

    PubMed Central

    Vega, Vinsensius B.; Thomsen, Jane S.; Kandhadayar, Gopalan Srinivasan; Ng, Patrick Wei Pern; Chiu, Kuo Ping; Pettersson, Sven; Wei, Chia Lin; Ruan, Yijun; Liu, Edison T.

    2009-01-01

    Background The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). PPARγ has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARγ have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARγ binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARγ binding sites in 3T3-L1 preadipocyte cells. Methodology/Principal Findings Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARγ and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARγ and RXR and that they are functionally capable of driving PPARγ specific transcription. Our results strongly indicate that PPARγ is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARγ/RXR association is enriched within the proximity of the 5′ region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARγ as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function

  19. Crystallographic location of two Zn{sup 2+} binding sites in the avian cytochrome bc1 complex

    SciTech Connect

    Berry, Edward A.; Zhang, Zhaolei; Bellamy, Henry D.; Huang, Lishar

    2000-06-20

    The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc1 complex) is inhibited by Zn{sup 2+} ions, but with higher Ki ({approximately}3 {micro}M) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 mM ZnCl{sub 2} for 7 days, the crystalline chicken bc1 complex specifically binds Zn{sup 2+} at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Qo of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Qo site and the bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c1, and might interfere with the egress of protons from the Qo site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions.

  20. Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

    PubMed Central

    Mgcina, Londiwe S.; Dubery, Ian A.; Piater, Lizelle A.

    2015-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies. PMID:26029233

  1. Opioid binding site in EL-4 thymoma cell line

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  2. Binding of dinitrogen to an iron-sulfur-carbon site

    NASA Astrophysics Data System (ADS)

    Čorić, Ilija; Mercado, Brandon Q.; Bill, Eckhard; Vinyard, David J.; Holland, Patrick L.

    2015-10-01

    Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

  3. Insights into the bile acid transportation system: the human ileal lipid-binding protein-cholyltaurine complex and its comparison with homologous structures.

    PubMed

    Kurz, Michael; Brachvogel, Volker; Matter, Hans; Stengelin, Siegfried; Thüring, Harald; Kramer, Werner

    2003-02-01

    Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three-dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid-binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP-cholylglycine complex and with related lipid-binding proteins. Protein structures were determined in solution by using two-dimensional (2D)- and 3D-homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine-binding site. The bile acid is deeply buried within ILBP with its flexible side-chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein-binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system. PMID:12486725

  4. Using Micropatterned Lipid Bilayer Arrays to Measure the Effect of Membrane Composition on Merocyanine 540 Binding

    PubMed Central

    Smith, Kathryn A.; Conboy, John C.

    2011-01-01

    The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 bindingin DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner. PMID:21376014

  5. Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli.

    PubMed

    Jordanova, Rositsa; Radoslavov, Georgi; Fischer, Peter; Liebau, Eva; Walter, Rolf D; Bankov, Ilia; Boteva, Raina

    2005-01-01

    Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids. PMID:15634342

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

    PubMed

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

    2016-08-25

    The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs. PMID:27565349

  7. Occupancy of the iron binding sites of human transferrin.

    PubMed Central

    Huebers, H A; Josephson, B; Huebers, E; Csiba, E; Finch, C A

    1984-01-01

    The in vivo distribution of iron between the binding sites of transferrin was examined. Plasma was obtained from normal subjects under basal conditions and after in vitro and in vivo iron loading. Independent methods, including measurement of the transferrin profile after isoelectric focusing and cross immunoelectrophoresis, and determination of the iron content in the separated fractions were in agreement that there was a random distribution of iron on binding sites. This held true with in vitro loading, when iron was increased by intestinal absorption and with loading from the reticuloendothelial system. The data indicate that the distribution of apo-, monoferric, and diferric transferrins is predictable on the basis of the plasma transferrin saturation and negate the concept that iron loading of transferrin in vitro is a selective process with possible functional consequences in tissue iron delivery. PMID:6589596

  8. The Allosteric Binding Sites of Sulfotransferase 1A1

    PubMed Central

    Cook, Ian; Wang, Ting; Falany, Charles N.

    2015-01-01

    Human sulfotransferases (SULTs) comprise a small, 13-member enzyme family that regulates the activities of thousands of compounds—endogenous metabolites, drugs, and other xenobiotics. SULTs transfer the sulfuryl-moiety (–SO3) from a nucleotide donor, PAPS (3′-phosphoadenosine 5′-phosphosulfate), to the hydroxyls and primary amines of acceptors. SULT1A1, a progenitor of the family, has evolved to sulfonate compounds that are remarkably structurally diverse. SULT1A1, which is found in many tissues, is the predominant SULT in liver, where it is a major component of phase II metabolism. Early work demonstrated that catechins and nonsteroidal anti-inflammatory drugs inhibit SULT1A1 and suggested that the inhibition was not competitive versus substrates. Here, the mechanism of inhibition of a single, high affinity representative from each class [epigallocatechin gallate (EGCG) and mefenamic acid] is determined using initial-rate and equilibrium-binding studies. The findings reveal that the inhibitors bind at sites separate from those of substrates, and at saturation turnover of the enzyme is reduced to a nonzero value. Further, the EGCG inhibition patterns suggest a molecular explanation for its isozyme specificity. Remarkably, the inhibitors bind at sites that are separate from one another, and binding at one site does not affect affinity at the other. For the first time, it is clear that SULT1A1 is allosterically regulated, and that it contains at least two, functionally distinct allosteric sites, each of which responds to a different class of compounds. PMID:25534770

  9. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  10. Ionic Hydrogen Bonds and Lipid Packing Defects Determine the Binding Orientation and Insertion Depth of RecA on Multicomponent Lipid Bilayers.

    PubMed

    Zhang, Leili; Rajendram, Manohary; Weibel, Douglas B; Yethiraj, Arun; Cui, Qiang

    2016-08-25

    We describe a computational and experimental approach for probing the binding properties of the RecA protein at the surface of anionic membranes. Fluorescence measurements indicate that RecA behaves differently when bound to phosphatidylglycerol (PG)- and cardiolipin (CL)-containing liposomes. We use a multistage computational protocol that integrates an implicit membrane/solvent model, the highly mobile mimetic membrane model, and the full atomistic membrane model to study how different anionic lipids perturb RecA binding to the membrane. With anionic lipids studied here, the binding interface involves three key regions: the N-terminal helix, the DNA binding loop L2, and the M-M7 region. The nature of binding involves both electrostatic interactions between cationic protein residues and lipid polar/charged groups and insertion of hydrophobic residues. The L2 loop contributes more to membrane insertion than the N-terminal helix. More subtle aspects of RecA-membrane interaction are influenced by specific properties of anionic lipids. Ionic hydrogen bonds between the carboxylate group in phosphatidylserine and several lysine residues in the C-terminal region of RecA stabilize the parallel (∥) binding orientation, which is not locally stable on PG- and CL-containing membranes despite similarity in the overall charge density. Lipid packing defects, which are more prevalent in the presence of conical lipids, are observed to enhance the insertion depth of hydrophobic motifs. The computational finding that RecA binds in a similar orientation to PG- and CL-containing membranes is consistent with the fact that PG alone is sufficient to induce RecA polar localization, although CL might be more effective because of its tighter binding to RecA. The different fluorescence behaviors of RecA upon binding to PG- and CL-containing liposomes is likely due to the different structures and flexibility of the C-terminal region of RecA when it binds to different anionic phospholipids

  11. DNA methylation presents distinct binding sites for human transcription factors.

    PubMed

    Hu, Shaohui; Wan, Jun; Su, Yijing; Song, Qifeng; Zeng, Yaxue; Nguyen, Ha Nam; Shin, Jaehoon; Cox, Eric; Rho, Hee Sool; Woodard, Crystal; Xia, Shuli; Liu, Shuang; Lyu, Huibin; Ming, Guo-Li; Wade, Herschel; Song, Hongjun; Qian, Jiang; Zhu, Heng

    2013-01-01

    DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001. PMID:24015356

  12. DNA methylation presents distinct binding sites for human transcription factors

    PubMed Central

    Hu, Shaohui; Wan, Jun; Su, Yijing; Song, Qifeng; Zeng, Yaxue; Nguyen, Ha Nam; Shin, Jaehoon; Cox, Eric; Rho, Hee Sool; Woodard, Crystal; Xia, Shuli; Liu, Shuang; Lyu, Huibin; Ming, Guo-Li; Wade, Herschel; Song, Hongjun; Qian, Jiang; Zhu, Heng

    2013-01-01

    DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI: http://dx.doi.org/10.7554/eLife.00726.001 PMID:24015356

  13. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

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

    2010-09-02

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

  14. Binding of Daptomycin to Anionic Lipid Vesicles Is Reduced in the Presence of Lysyl-Phosphatidylglycerol.

    PubMed

    Khatib, Tala O; Stevenson, Heather; Yeaman, Michael R; Bayer, Arnold S; Pokorny, Antje

    2016-08-01

    The cytoplasmic membrane of Staphylococcus aureus contains ∼20 mol% of the net cationic lipid lysyl-phosphatidylglycerol (LPG). Elevated fractions of LPG are associated with increased resistance to cationic antibiotics, including the lipopeptide daptomycin (DAP). Although the surface charge of the bacterial cytoplasmic membrane is altered by LPG, surface binding of DAP was found to be only moderately affected in anionic vesicles containing 20 mol% LPG. These results suggest that charge repulsion cannot fully explain LPG-mediated resistance to cationic peptides. PMID:27216066

  15. 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

  16. 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

  17. Binding characterization, synthesis and biological evaluation of RXRα antagonists targeting the coactivator binding site.

    PubMed

    Xu, Dingyu; Guo, Shangjie; Chen, Ziwen; Bao, Yuzhou; Huang, Fengyu; Xu, Dan; Zhang, Xindao; Zeng, Zhiping; Zhou, Hu; Zhang, Xiaokun; Su, Ying

    2016-08-15

    Previously we identified the first retinoid X receptor-alpha (RXRα) modulators that regulate the RXRα biological function via binding to the coregulator-binding site. Here we report the characterization of the interactions between the hit molecule and RXRα through computational modeling, mutagenesis, SAR and biological evaluation. In addition, we reported studies of additional new compounds and identified a molecule that mediated the NF-κB pathway by inhibiting the TNFα-induced IκBα degradation and p65 nuclear translocation. PMID:27450787

  18. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    PubMed Central

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-01-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL. PMID:25737239

  19. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    DOE PAGESBeta

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; et al

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobicmore » environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.« less

  20. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    SciTech Connect

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  1. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  2. Roles played by acidic lipids in HIV-1 Gag membrane binding

    PubMed Central

    Olety, Balaji; Ono, Akira

    2014-01-01

    The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids. PMID:24998886

  3. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics.

    PubMed

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  4. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    PubMed Central

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  5. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    NASA Astrophysics Data System (ADS)

    Woo, Sun Young; Lee, Hwankyu

    2016-03-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

  6. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

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

    SciTech Connect

    Lummis, S.C.R.; Johnston, G.A.R. ); Nicoletti, G. ); Holan, G. )

    1991-01-01

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

  8. Identification and Characterization of the Lipid-Binding Property of GrlR, a Locus of Enterocyte Effacement Regulator

    SciTech Connect

    Jobichen, C.; Fernandis, A; Velazquez-Campoy, A; Leung, K; Mok, Y; Wenk, M; Sivaraman, J

    2009-01-01

    Lipocalins are a broad family of proteins identified initially in eukaryotes and more recently in Gram-negative bacteria. The functions of lipocalin or lipid-binding proteins are often elusive and very diverse. Recently, we have determined the structure of GrlR (global regulator of LEE repressor), which plays a key role in the regulation of LEE (locus of enterocyte effacement) proteins. GrlR adopts a lipocalin-like fold that is composed of an eight-stranded e-barrel followed by an a-helix at the C-terminus. GrlR has a highly hydrophobic cavity region and could be a potential transporter of lipophilic molecules. To verify this hypothesis, we carried out structure-based analysis of GrlR, determined the structure of the lipid-GrlR complex and measured the binding of lipid to recombinant GrlR by ITC (isothermal titration calorimetry). In addition, we identified phosphatidylglycerol and phosphatidylethanolamine as the endogenously bound lipid species of GrlR using electrospray-ionization MS. Furthermore, we have shown that the lipid-binding property of GrlR is similar to that of its closest lipocalin structural homologue, e-lactoglobulin. Our studies demonstrate the hitherto unknown lipid-binding property of GrlR.

  9. Stable, ligand-doped, poly(bis-SorbPC) lipid bilayer arrays for protein binding and detection.

    PubMed

    Joubert, James R; Smith, Kathryn A; Johnson, Erin; Keogh, John P; Wysocki, Vicki H; Gale, Bruce K; Conboy, John C; Saavedra, S Scott

    2009-06-01

    A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM(1), and the extent of binding was correlated to the mole percentage of GM(1) in each spot. A poly(lipid) bilayer array composed of spots doped with GM(1) and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM(1) arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies. PMID:20355927

  10. All-Purpose Containers? Lipid-Binding Protein – Drug Interactions

    PubMed Central

    Beringhelli, Tiziana; Gianazza, Elisabetta; Maggioni, Daniela; Scanu, Sandra; Parravicini, Chiara; Sensi, Cristina; Monaco, Hugo L.; Eberini, Ivano

    2015-01-01

    The combined use of in vitro (19F-NMR) and in silico (molecular docking) procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine) for different drugs (mainly steroids and vastatins). Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding). Dissociation constants (Ki) for drugs typically lie in the same range as Ki for natural ligands; in most instances (different proteins and docking conditions), vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues). PMID:26167932

  11. Lipid composition is important for highly efficient target binding and retention of immunoliposomes

    SciTech Connect

    Maruyama, Kazuo; Huang, L. ); Kennel, S.J. )

    1990-08-01

    By taking advantage of a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, the authors have prepared liposomes containing various amounts of antibody molecules (immunoliposomes). These immunoliposomes accumulate specifically in the lung when injected i.v. Two lipid compositions were used: phosphatidylcholine/cholesterol/phosphatidylserine (PS), 10:5:1 (mol/mol), a composition that allows liposomes to be readily taken up by the reticuloendothelial system (RES) (liver and spleen), and phosphatidylcholine/cholesterol/ganglioside GM{sub 1}, 10:5:1 (mol/mol), a composition that allows liposomes to avoid or delay the RES uptake (the so-called stealth liposomes). Although an increase in the number of antibody molecules per liposome was accompanied by an increased level of lung binding of the immunoliposomes, differences due to the lipid composition were more profound. Conjugation of antibody to the stealth liposome did not increase the rate of liposome uptake by liver; this rate was approximately 10-fold lower than that of the PS-containing liposomes without antibody. Stealth immunoliposomes with high antibody content also showed long retention in the lung. These results can be understood on the basis of two competing kinetic processes: lung binding whose rate is directly proportional to the antibody content of the immunoliposomes and uptake by RES whose rate is significantly reduced in the case of the stealth liposomes.

  12. Anionic lipid binding to the foreign protein MGS provides a tight coupling between phospholipid synthesis and protein overexpression in Escherichia coli.

    PubMed

    Ariöz, Candan; Ye, Weihua; Bakali, Amin; Ge, Changrong; Liebau, Jobst; Götzke, Hansjörg; Barth, Andreas; Wieslander, Ake; Mäler, Lena

    2013-08-20

    Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by (31)P NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a "feed-forward" stimulation of lipid synthesis in E. coli. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis. PMID:23869703

  13. Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel

    PubMed Central

    Martin, Lewis J.; Corry, Ben

    2014-01-01

    Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites. PMID:24992293

  14. Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site.

    PubMed

    Oikonomakos, N G; Schnier, J B; Zographos, S E; Skamnaki, V T; Tsitsanou, K E; Johnson, L N

    2000-11-01

    Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes. PMID:10924512

  15. PeptiSite: a structural database of peptide binding sites in 4D.

    PubMed

    Acharya, Chayan; Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2014-03-21

    We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include: (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a multimeric protein assembly with cofactors and metal ions and (3) analysis of consensus interaction motifs within the ensembles and identification of conserved determinants of these interactions. Currently the database contains 585 proteins with 650 peptide-binding sites. http://peptisite.ucsd.edu/ link allows searching for the sites of interest and interactive visualization of the ensembles using the ActiveICM web-browser plugin. This structural database for protein-peptide interactions enables understanding of structural principles of these interactions and may assist the development of an efficient peptide docking benchmark. PMID:24406170

  16. Sex hormone-binding globulin and lipid profile in pubertal children.

    PubMed

    Garcés, Carmen; Oya, Iría de; Lasunción, Miguel A; López-Simón, Laura; Cano, Beatriz; de Oya, Manuel

    2010-02-01

    Men and women have different lipid profiles throughout life, related to changes in sex hormones; and this has been associated with sex-related differences in the prevalence of coronary heart disease. The influence of sex hormone changes during puberty on the lipid profile has been reported, but levels of sex hormone-binding globulin (SHBG) (the specific plasma binding protein of sex hormones) have not been evaluated even though its regulatory role might be crucial. The aim of this study was to analyze the relationship between sex hormones and SHBG and changes in plasma lipid levels during puberty. Our population-based sample included 370 healthy schoolchildren (175 male and 195 female), ranging from 12 to 15 years old. High-density lipoprotein cholesterol (HDL-C) levels were significantly lower in 15-year-olds than in younger boys, and apolipoprotein (apo) A-I levels steeply decreased across the studied age groups. Parallel to these changes, testosterone levels increased whereas SHBG decreased as age increases in boys. In girls, no significant differences were observed in these variables among the age groups. Testosterone and SHBG were highly correlated with anthropometric variables. Sex hormone-binding globulin was negatively associated with triglycerides (TG) in both sexes, remaining statistically significant after further adjustment for age and body mass index (BMI) in girls. Sex hormone-binding globulin was the only predictive variable for HDL-C and TG in multiple linear regression analysis, after adjustment by BMI, in both sexes, accounting for 10% of the variance of HDL-C in boys and for around 5% of the variance of TG in both sexes. In boys, testosterone and SHBG remained significantly correlated to apo A-I levels, even after adjusting for age and BMI, and were the most important predictive variables for apo A-I in multiple linear regression analysis. In conclusion, SHBG levels are related to a decrease in HDL-C and apo A-I levels during puberty in boys and

  17. Detection and quantification of lipid membrane binding on silica micro-tube resonator sensor

    NASA Astrophysics Data System (ADS)

    Ling, Tao; Majd, Sheereen; Mayer, Michael; Guo, L. Jay

    2008-02-01

    Silica micro-tube resonator is a very attractive biosensor platform for label-free detection of bio-molecules by combining high sensitivity and simple fluidic handling. We report the study of lipid membrane binding on micro-tube sensor that is probed by a prism coupling technique. Prism coupling to the micro-tube resonance modes allows thick micro-tube to be used and the selectivity of high-sensitivity modes. We were able to identify and probe a special resonance mode that has very high Electrical field at the boundary of the fluid and the inner tube wall. Unlike typical WGM modes, such resonance modes also have very high Electrical field extending into the lower index fluid region, thereby providing exceptionally high sensitivity to the fluid's refractive index change. We used this type of resonance mode to detect the formation of single bilayer lipid membrane on the inner tube wall. With 4-5nm POPC lipid membrane with refractive index around 1.46 absorbed on the inner wall, we can observe the resonance peak shift around 44pm. Mie scattering simulation of the resonance peak shift due to the bio-film absorbed onto the inner wall agrees very well with the experiment results. We also observed resonance peak shift with the membrane protein Annexin V bonding to the lipid membrane. With the present Q factor 6×10 4 at 1.55μm wavelength, we estimate that our devices can detect the presence of 0.1nm thick absorbed film on the inner wall of the tube. The device's sensitivity can be greatly enhanced by switching the working wavelength from infrared wavelength to visible wavelength where water absorption is minimized.

  18. Positional distribution of transcription factor binding sites in Arabidopsis thaliana

    PubMed Central

    Yu, Chun-Ping; Lin, Jinn-Jy; Li, Wen-Hsiung

    2016-01-01

    Binding of a transcription factor (TF) to its DNA binding sites (TFBSs) is a critical step to initiate the transcription of its target genes. It is therefore interesting to know where the TFBSs of a gene are likely to locate in the promoter region. Here we studied the positional distribution of TFBSs in Arabidopsis thaliana, for which many known TFBSs are now available. We developed a method to identify the locations of TFBSs in the promoter sequences of genes in A. thaliana. We found that the distribution is nearly bell-shaped with a peak at 50 base pairs (bp) upstream of the transcription start site (TSS) and 86% of the TFBSs are in the region from −1,000 bp to +200 bp with respect to the TSS. Our distribution was supported by chromatin immunoprecipitation sequencing and microarray data and DNase I hypersensitive site sequencing data. When TF families were considered separately, differences in positional preference were observed between TF families. Our study of the positional distribution of TFBSs seems to be the first in a plant. PMID:27117388

  19. Structural neighboring property for identifying protein-protein binding sites

    PubMed Central

    2015-01-01

    Background The protein-protein interaction plays a key role in the control of many biological functions, such as drug design and functional analysis. Determination of binding sites is widely applied in molecular biology research. Therefore, many efficient methods have been developed for identifying binding sites. In this paper, we calculate structural neighboring property through Voronoi diagram. Using 6,438 complexes, we study local biases of structural neighboring property on interface. Results We propose a novel statistical method to extract interacting residues, and interacting patches can be clustered as predicted interface residues. In addition, structural neighboring property can be adopted to construct a new energy function, for evaluating docking solutions. It includes new statistical property as well as existing energy items. Comparing to existing methods, our approach improves overall Fnat value by at least 3%. On Benchmark v4.0, our method has average Irmsd value of 3.31Å and overall Fnat value of 63%, which improves upon Irmsd of 3.89 Å and Fnat of 49% for ZRANK, and Irmsd of 3.99Å and Fnat of 46% for ClusPro. On the CAPRI targets, our method has average Irmsd value of 3.46 Å and overall Fnat value of 45%, which improves upon Irmsd of 4.18 Å and Fnat of 40% for ZRANK, and Irmsd of 5.12 Å and Fnat of 32% for ClusPro. Conclusions Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein binding sites, with the prediction quality improved in terms of CAPRI evaluation criteria. PMID:26356630

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

    SciTech Connect

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

    2004-10-28

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

  1. Cationic Cell-Penetrating Peptide Binds to Planar Lipid Bilayers Containing Negatively Charged Lipids but does not Induce Conductive Pores

    PubMed Central

    Gurnev, Philip A.; Yang, Sung-Tae; Melikov, Kamran C.; Chernomordik, Leonid V.; Bezrukov, Sergey M.

    2013-01-01

    Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 μM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 μM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes. PMID:23663836

  2. Fat(al) attraction: Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles.

    PubMed

    van der Schaar, Hilde M; Dorobantu, Cristina M; Albulescu, Lucian; Strating, Jeroen R P M; van Kuppeveld, Frank J M

    2016-07-01

    All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome. Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. While some lipids may be locally synthesized de novo, other lipids are shuttled towards ROs. In picornavirus-infected cells, lipids are exchanged at membrane contact sites between ROs and other organelles. In this paper, we review recent advances in our understanding of how picornaviruses exploit host membrane contact site machinery to generate ROs, a mechanism that is used by some other +RNA viruses as well. PMID:27020598

  3. CryptoSite: Expanding the Druggable Proteome by Characterization and Prediction of Cryptic Binding Sites.

    PubMed

    Cimermancic, Peter; Weinkam, Patrick; Rettenmaier, T Justin; Bichmann, Leon; Keedy, Daniel A; Woldeyes, Rahel A; Schneidman-Duhovny, Dina; Demerdash, Omar N; Mitchell, Julie C; Wells, James A; Fraser, James S; Sali, Andrej

    2016-02-22

    Many proteins have small-molecule binding pockets that are not easily detectable in the ligand-free structures. These cryptic sites require a conformational change to become apparent; a cryptic site can therefore be defined as a site that forms a pocket in a holo structure, but not in the apo structure. Because many proteins appear to lack druggable pockets, understanding and accurately identifying cryptic sites could expand the set of drug targets. Previously, cryptic sites were identified experimentally by fragment-based ligand discovery and computationally by long molecular dynamics simulations and fragment docking. Here, we begin by constructing a set of structurally defined apo-holo pairs with cryptic sites. Next, we comprehensively characterize the cryptic sites in terms of their sequence, structure, and dynamics attributes. We find that cryptic sites tend to be as conserved in evolution as traditional binding pockets but are less hydrophobic and more flexible. Relying on this characterization, we use machine learning to predict cryptic sites with relatively high accuracy (for our benchmark, the true positive and false positive rates are 73% and 29%, respectively). We then predict cryptic sites in the entire structurally characterized human proteome (11,201 structures, covering 23% of all residues in the proteome). CryptoSite increases the size of the potentially "druggable" human proteome from ~40% to ~78% of disease-associated proteins. Finally, to demonstrate the utility of our approach in practice, we experimentally validate a cryptic site in protein tyrosine phosphatase 1B using a covalent ligand and NMR spectroscopy. The CryptoSite Web server is available at http://salilab.org/cryptosite. PMID:26854760

  4. Identification and in silico analysis of helical lipid binding regions in proteins belonging to the amphitropic protein family.

    PubMed

    Keller, Rob C A

    2014-12-01

    The role of protein-lipid interactions is increasingly recognized to be of importance in numerous biological processes. Bioinformatics is being increasingly used as a helpful tool in studying protein-lipid interactions. Especially recently developed approaches recognizing lipid binding regions in proteins can be implemented. In this study one of those bioinformatics approaches specialized in identifying lipid binding helical regions in proteins is expanded. The approach is explored further by features which can be easily obtained manually. Some interesting examples of members of the amphitropic protein family have been investigated in order to demonstrate the additional features of this bioinformatics approach. The results in this study seem to indicate interesting characteristics of amphitropic proteins and provide insight into the mechanistic functioning and overall understanding of this intriguing class of proteins. Additionally, the results demonstrate that the presented bioinformatics approach might be either an interesting starting point in protein-lipid interactions studies or a good tool for selecting new focus points for more detailed experimental research of proteins with known overall protein-lipid binding abilities. PMID:25431407

  5. Membrane binding of peptide models for early stages of amyloid formation: Lipid packing counts more than charge.

    PubMed

    Hoernke, Maria; Tassler, Stephanie; Koksch, Beate; Brezesinski, Gerald

    2016-06-01

    Amyloid formation is related to neurodegenerative diseases like Alzheimer's disease or Parkinson's disease. In the molecular onset of the diseases, soluble peptides adopt conformations that are rich in β-sheet and ultimately form aggregates. How this process is triggered or influenced by membrane binding, or how the membrane integrity is disturbed by the peptide binding and conformational transition is still under debate. In the present study, we systematically examine the effects of β-sheet prone model peptides on zwitterionic and negatively charged lipids in both mono- and bilayers and in various lipid phase states by infrared reflection absorption spectroscopy, grazing incidence X-ray diffraction, and small and wide angle X-ray scattering. No difference in the interaction of the peptides with zwitterionic or negatively charged lipids was observed. Furthermore, the interaction of β-sheet prone model peptides leaves the lipid structure largely unaffected. However, the lipid phase state decides upon the mode of interaction. Peptides insert into liquid-expanded layers and interact only with the head groups of liquid-condensed lipid layers. Using a zoo of complementary techniques and critically examining preparation procedures we are able to obtain an unambiguous picture of peptide binding to membranes. PMID:27134131

  6. Semisynthetic Lipopeptides Derived from Nisin Display Antibacterial Activity and Lipid II Binding on Par with That of the Parent Compound.

    PubMed

    Koopmans, Timo; Wood, Thomas M; 't Hart, Peter; Kleijn, Laurens H J; Hendrickx, Antoni P A; Willems, Rob J L; Breukink, Eefjan; Martin, Nathaniel I

    2015-07-29

    The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic constructs that display potent inhibition of bacterial growth, with activities approaching that of nisin itself. Most notable was the activity observed against clinically relevant bacterial strains including MRSA and VRE. Experiments with membrane models indicate that these constructs operate via a lipid II-mediated mode of action without causing pore formation. A lipid II-dependent mechanism of action is further supported by antagonization assays wherein the addition of lipid II was found to effectively block the antibacterial activity of the nisin-derived lipopeptides. PMID:26122963

  7. Viral receptor-binding site antibodies with diverse germline origins

    PubMed Central

    Schmidt, Aaron G.; Therkelsen, Matthew D.; Stewart, Shaun; Kepler, Thomas B.; Liao, Hua-Xin; Moody, M. Anthony; Haynes, Barton F.; Harrison, Stephen C.

    2015-01-01

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by eleven different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B-cell targets. PMID:25959776

  8. Viral receptor-binding site antibodies with diverse germline origins.

    PubMed

    Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

    2015-05-21

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. PMID:25959776

  9. Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

    PubMed

    Potvin-Fournier, Kim; Lefèvre, Thierry; Picard-Lafond, Audrey; Marcotte, Catherine; Dufresne, Caroline; Cantin, Line; Salesse, Christian; Auger, Michèle

    2016-06-21

    Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium. PMID:27240971

  10. Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin.

    PubMed

    Gross, Danielle N; Miyoshi, Hideaki; Hosaka, Toshio; Zhang, Hui-Hong; Pino, Elizabeth C; Souza, Sandra; Obin, Martin; Greenberg, Andrew S; Pilch, Paul F

    2006-02-01

    In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure. PMID:16239256

  11. Analysis of Binding at a Single Spatially Localized Cluster of Binding Sites by Fluorescence Recovery after Photobleaching

    PubMed Central

    Sprague, Brian L.; Müller, Florian; Pego, Robert L.; Bungay, Peter M.; Stavreva, Diana A.; McNally, James G.

    2006-01-01

    Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed in live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a three-dimensional FRAP model that accounts for a single spatially localized cluster of binding sites in the presence of both diffusion and impermeable boundaries. We demonstrate that models completely ignoring the spatial localization of binding yield poor estimates for the binding parameters within the binding site cluster. In contrast, we find that ignoring only the restricted axial height of the binding-site cluster is far less detrimental, thereby enabling the use of computationally less expensive models. We also identify simplified solutions to the FRAP model for limiting behaviors where either diffusion or binding dominate. We show how ignoring a role for diffusion can sometimes produce serious errors in binding parameter estimation. We illustrate application of the method by analyzing binding of a transcription factor, the glucocorticoid receptor, to a tandem array of mouse mammary tumor virus promoter sites in live cells, obtaining an estimate for an in vivo binding constant (10−7 M), and a first approximation of an upper bound on the transcription-factor residence time at the promoter (∼170 ms). These FRAP analysis tools will be important for measuring key cellular binding parameters necessary for a complete and accurate description of the networks that regulate cellular behavior. PMID:16679358

  12. Atrial natriuretic factor binding sites in experimental congestive heart failure

    SciTech Connect

    Bianchi, C.; Thibault, G.; Wrobel-Konrad, E.; De Lean, A.; Genest, J.; Cantin, M. )

    1989-10-01

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor ((Ser99, Tyr126)ANF) binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF (des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2) (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease.

  13. Conserved properties of individual Ca2+-binding sites in calmodulin

    PubMed Central

    Halling, D. Brent; Liebeskind, Benjamin J.; Hall, Amelia W.; Aldrich, Richard W.

    2016-01-01

    Calmodulin (CaM) is a Ca2+-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca2+-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca2+-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca2+ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease. PMID:26884197

  14. 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

  15. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  16. AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain.

    PubMed Central

    Welters, P; Takegawa, K; Emr, S D; Chrispeels, M J

    1994-01-01

    The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110). The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus. The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant. PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase. Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development. Images PMID:7972072

  17. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

    SciTech Connect

    Harada, Y.; Li, H.; Li, Hua; Lennarz, W. J.

    2009-04-28

    Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.

  18. The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus.

    PubMed

    Meier, Christoph; Aricescu, A Radu; Assenberg, Rene; Aplin, Robin T; Gilbert, Robert J C; Grimes, Jonathan M; Stuart, David I

    2006-07-01

    To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds. PMID:16843897

  19. Fluorine Bonding Enhances the Energetics of Protein-Lipid Binding in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Liu, Lan; Jalili, Nobar; Baergen, Alyson; Ng, Simon; Bailey, Justin; Derda, Ratmir; Klassen, John S.

    2014-05-01

    This paper reports on the first experimental study of the energies of noncovalent fluorine bonding in a protein-ligand complex in the absence of solvent. Arrhenius parameters were measured for the dissociation of gaseous deprotonated ions of complexes of bovine β-lactoglobulin (Lg), a model lipid-binding protein, and four fluorinated analogs of stearic acid (SA), which contained (X =) 13, 15, 17, or 21 fluorine atoms. In all cases, the activation energies (Ea) measured for the loss of neutral XF-SA from the (Lg + XF-SA)7- ions are larger than for SA. From the kinetic data, the average contribution of each > CF2 group to Ea was found to be ~1.1 kcal mol-1, which is larger than the ~0.8 kcal mol-1 value reported for > CH2 groups. Based on these results, it is proposed that fluorocarbon-protein interactions are inherently stronger (enthalpically) than the corresponding hydrocarbon interactions.

  20. Binding sites for interaction of peroxiredoxin 6 with surfactant protein A.

    PubMed

    Krishnaiah, Saikumari Y; Dodia, Chandra; Sorokina, Elena M; Li, Haitao; Feinstein, Sheldon I; Fisher, Aron B

    2016-04-01

    Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H(+)-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell. PMID:26723227

  1. Lipid-Linked Oligosaccharides in Membranes Sample Conformations That Facilitate Binding to Oligosaccharyltransferase

    PubMed Central

    Kern, Nathan R.; Lee, Hui Sun; Wu, Emilia L.; Park, Soohyung; Vanommeslaeghe, Kenno; MacKerell, Alexander D.; Klauda, Jeffery B.; Jo, Sunhwan; Im, Wonpil

    2014-01-01

    Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs’ preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST. PMID:25418169

  2. The Use ofa Hydrophobic Binding Peptide Modified Lipid Nanocarrier Improving Tumor Distribution and Antitumor Efficacy.

    PubMed

    Gao, Wei; Yang, Xiucong; Lin, Zhiqiang; Gao, Shanyun; He, Bing; Mei, Bong; Wang, Dan; Yuan, Lan; Zhang, Hua; Dai, Wenbing; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2016-06-01

    In addition to showing the specific interaction between a generalized ligand and its receptor and the electrostatic effect between positive cell-penetrating peptides and negative cell membranes, our last study demonstrated the hydrophobic interactivity between a hydrophobic binding peptide (HBP) and biomembranes to be favorable in drug delivery. To yield more evidence for this new strategy and to find more effective HBPs, here we designed and established a novel nanomedicine associated with cyclosporin A (CsA) because this peptide is electrically neutral, highly hydrophobic, very stable in vivo and safe at the given dose. First, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) studies showed a strong hydrophobic interaction between the CsA molecules and the lipid membrane. The lactate dehydrogenase release assay proved that CsA exhibited low toxicity to cell membranes. These facts encouraged us to explore the potential application of CsA as an HBP to actualize intracellular delivery of nanomedicines for tumor therapy. When conjugated to lipid nanocarriers, CsA significantly enhanced their binding with cells and,. consequently, increased the internalization of recoded nanomedicines into cells. The in vivo experiments further showed that the CsA-associated nanocarriers could achieve better delivery to tumor tissues and improve the tumor therapy of doxorubicin (DOX) compared to the nonmodified control; these findings were identical to the observations-in cell studies. In conclusion, CsA, a readily obtainable molecule with favorable characteristics, is indeed a good candidate for an HBP, and this study provides solid, novel evidence for the use of HBP-based nanocarriers as effective antitumor drug delivery systems. PMID:27319213

  3. NMR Mapping of the IFNAR1-EC binding site on IFNα2 reveals allosteric changes in the IFNAR2-EC binding site

    PubMed Central

    Akabayov, Sabine Ruth; Biron, Zohar; Lamken, Peter; Piehler, Jacob; Anglister, Jacob

    2010-01-01

    All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits IFNAR1 and IFNAR2. In this study we mapped the surface of IFNα2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the [1H,15N]-TROSY-HSQC cross peaks of IFNα2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNα2 with IFNAR2-EC. The NMR study on the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 out of 165 IFNα2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNα2 binding site for IFNAR2-EC indicating allosteric communication between the binding sites for the two receptor subunits. PMID:20047337

  4. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

    PubMed Central

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

  5. The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

    PubMed

    Kanno, Takeshi; Yamamoto, Hideyuki; Yaguchi, Takahiro; Hi, Rika; Mukasa, Takeshi; Fujikawa, Hirokazu; Nagata, Tetsu; Yamamoto, Satoshi; Tanaka, Akito; Nishizaki, Tomoyuki

    2006-06-01

    This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation. PMID:16520488

  6. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    SciTech Connect

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-11-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for /sup 125/I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.

  7. Effect of dietary cellulose on site of lipid absorption

    SciTech Connect

    Gallaher, D.; Schneeman, B.O.

    1985-08-01

    The effect of dietary cellulose on the localization within the small intestine of isotopically labeled triglyceride (TG) and cholesterol (CH) from a test meal was investigated. Feeding a 20% cellulose meal resulted in greater quantities of /sup 14/C-TG present in both the contents and mucosa of the distal intestine compared with a fiber-free control meal. In contrast, cellulose had no effect on the localization of CH within either the intestinal contents or the mucosa. Accumulation of TG within the intestine was not due to differences in stomach emptying, as the emptying rate was similar for both TG and CH. Within the bulk phase TG must be hydrolyzed by pancreatic lipase before it is available for cellular uptake at the microvillus membrane, whereas CH requires no hydrolysis. The greater amount of TG, but not of CH, within the intestine suggests that cellulose can interfere with lipase activity in vivo. Consequently, cellulose can delay TG hydrolysis and increase the amount of lipid absorbed in the ileum.

  8. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Zhao, Guangyu; Chen, Shoucong; Liu, Feng; Sun, Ying; Zhang, Li

    2009-07-01

    The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants ( Ka) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M -1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be -3.634 kJ mol -1 and 72.66 J mol -1 K -1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed.

  9. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  10. Lipid binding specificity of bovine α-lactalbumin: a multidimensional approach.

    PubMed

    Chaudhuri, Arunima; Chattopadhyay, Amitabha

    2014-08-01

    Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA-membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA-oleic acid complex. PMID:24802274

  11. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

  12. The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

    PubMed Central

    Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

    2011-01-01

    Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

  13. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.

  14. XAS and Pulsed EPR Studies of the Copper Binding Site in Riboflavin Binding Protein

    SciTech Connect

    Smith,S.; Bencze, K.; Wasiukanis, K.; Benore-Parsons, T.; Stemmler, T.

    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 Angstroms, 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 CuO3N 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.

  15. POBO, transcription factor binding site verification with bootstrapping

    PubMed Central

    Kankainen, Matti; Holm, Liisa

    2004-01-01

    Transcription factors can either activate or repress target genes by binding onto short nucleotide sequence motifs in the promoter regions of these genes. Here, we present POBO, a promoter bootstrapping program, for gene expression data. POBO can be used to detect, compare and verify predetermined transcription factor binding site motifs in the promoters of one or two clusters of co-regulated genes. The program calculates the frequencies of the motif in the input promoter sets. A bootstrap analysis detects significantly over- or underrepresented motifs. The output of the program presents bootstrapped results in picture and text formats. The program was tested with published data from transgenic WRKY70 microarray experiments. Intriguingly, motifs recognized by the WRKY transcription factors of plant defense pathways are similarly enriched in both up- and downregulated clusters. POBO analysis suggests slightly modified hypothetical motifs that discriminate between up- and downregulated clusters. In conclusion, POBO allows easy, fast and accurate verification of putative regulatory motifs. The statistical tests implemented in POBO can be useful in eliminating false positives from the results of pattern discovery programs and increasing the reliability of true positives. POBO is freely available from http://ekhidna.biocenter.helsinki.fi:9801/pobo. PMID:15215385

  16. 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.

  17. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

    Frank, Martin; Hähnlein-Schick, Irmgard; Ekström, Jens-Ola; Arnberg, Niklas; Stehle, Thilo

    2014-01-01

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC. PMID:25329320

  18. Single prenyl-binding site on protein prenyl transferases

    PubMed Central

    Desnoyers, Luc; Seabra, Miguel C.

    1998-01-01

    Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions. PMID:9770475

  19. Mapping protein binding sites on the biomolecular corona of nanoparticles

    NASA Astrophysics Data System (ADS)

    Kelly, Philip M.; Åberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetić, Željka; Dawson, Kenneth A.

    2015-05-01

    Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized ‘corona’ of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

  20. Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

    PubMed

    Whitfield, D M; Choay, J; Sarkar, B

    1992-06-01

    As model compounds for Ni(II)-binding heparin-like compounds isolated from human kidneys (Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two disaccharides--4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-D-mannitol, trisodium salt (1b)--that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high-performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous-acid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn (OAc)2 were analyzed to yield 1:1 Zn(II)-binding constants of 472 +/- 59, 698 +/- 120, 8,758 +/- 2,237 and 20,100 +/- 5,598 M-1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions. PMID:1643264

  1. Lipid Classes and Fatty Acids in Ophryotrocha cyclops, a Dorvilleid from Newfoundland Aquaculture Sites

    PubMed Central

    Salvo, Flora; Dufour, Suzanne C.; Hamoutene, Dounia; Parrish, Christopher C.

    2015-01-01

    A new opportunistic annelid (Ophryotrocha cyclops) discovered on benthic substrates underneath finfish aquaculture sites in Newfoundland (NL) may be involved in the remediation of organic wastes. At those aquaculture sites, bacterial mats and O. cyclops often coexist and are used as indicators of organic enrichment. Little is known on the trophic strategies used by these annelids, including whether they might consume bacteria or other aquaculture-derived wastes. We studied the lipid and fatty acid composition of the annelids and their potential food sources (degraded flocculent organic matter, fresh fish pellets and bacterial mats) to investigate feeding relationships in these habitats and compared the lipid and fatty acid composition of annelids before and after starvation. Fish pellets were rich in lipids, mainly terrestrially derived C18 fatty acids (18:1ω9, 18:2ω6, 18:3ω3), while bacterial samples were mainly composed of ω7 fatty acids, and flocculent matter appeared to be a mixture of fresh and degrading fish pellets, feces and bacteria. Ophryotrocha cyclops did not appear to store excessive amounts of lipids (13%) but showed a high concentration of ω3 and ω6 fatty acids, as well as a high proportion of the main fatty acids contained in fresh fish pellets and bacterial mats. The dorvilleids and all potential food sources differed significantly in their lipid and fatty acid composition. Interestingly, while all food sources contained low proportions of 20:5ω3 and 20:2ω6, the annelids showed high concentrations of these two fatty acids, along with 20:4ω6. A starvation period of 13 days did not result in a major decrease in total lipid content; however, microscopic observations revealed that very few visible lipid droplets remained in the gut epithelium after three months of starvation. Ophryotrocha cyclops appears well adapted to extreme environments and may rely on lipid-rich organic matter for survival and dispersal in cold environments. PMID:26308719

  2. An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus.

    PubMed

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Moncharmont, B; Molinari, A M; Puca, G A

    1990-07-31

    It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site. PMID:1696480

  3. Physicochemical features of the HERG channel drug binding site.

    PubMed

    Fernandez, David; Ghanta, Azad; Kauffman, Gregory W; Sanguinetti, Michael C

    2004-03-12

    Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. PMID:14699101

  4. A vertebrate model for the study of lipid binding/transfer protein function: conservation of OSBP-related proteins between zebrafish and human.

    PubMed

    Zhou, You; Wohlfahrt, Gerd; Paavola, Jere; Olkkonen, Vesa M

    2014-04-11

    Oxysterol-binding protein (OSBP) and OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a family of lipid-binding/transfer proteins (LTPs) present in eukaryotes from yeast to man. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and act as either lipid transporters or sensors that control lipid metabolism, cell signaling, and vesicle transport. Zebrafish, Danio rerio, has gained increasing popularity as a model organism in developmental biology, human disease, toxicology, and drug discovery. However, LTPs in the fish are thus far unexplored. In this article we report a series of bioinformatic analyses showing that the OSBPL gene family is highly conserved between the fish and human. The OSBPL subfamily structure is markedly similar between the two organisms, and all 12 human genes have orthologs, designated osbpl and located on 11 chromosomes in D. rerio. Interestingly, osbpl2 and osbpl3 are present as two closely related homologs (a and b), due to gene duplication events in the teleost lineage. Moreover, the domain structures of the distinct ORP proteins are almost identical between zebrafish and man, and molecular modeling in the present study suggests that ORD liganding by phosphatidylinositol-4-phosphate (PI4P) is a feature conserved between yeast Osh3p, human ORP3, and zebrafish Osbpl3. The present analysis identifies D. rerio as an attractive model to study the functions of ORPs in vertebrate development and metabolism. PMID:24326072

  5. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  6. Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

    PubMed Central

    Barajas, Daniel; Xu, Kai; de Castro Martín, Isabel Fernández; Sasvari, Zsuzsanna; Brandizzi, Federica; Risco, Cristina; Nagy, Peter D.

    2014-01-01

    Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication. PMID:25329172

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

    NASA Astrophysics Data System (ADS)

    Clifford, Jacob; Adami, Christoph

    2015-10-01

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

  8. High-affinity dextromethorphan binding sites in guinea pig brain. II. Competition experiments.

    PubMed

    Craviso, G L; Musacchio, J M

    1983-05-01

    Binding of dextromethorphan (DM) to guinea pig brain is stereoselective, since levomethorphan is 20 times weaker than DM in competing for DM sites. In general, opiate agonists and antagonists as well as their corresponding dextrorotatory isomers are weak competitors for tritiated dextromethorphan ([3H]DM) binding sites and display IC50 values in the micromolar range. In contrast, several non-narcotic, centrally acting antitussives are inhibitory in the nanomolar range (IC50 values for caramiphen, carbetapentane, dimethoxanate, and pipazethate are 25 nM, 9 nM, 41 nM, and 190 nM, respectively). Other antitussives, such as levopropoxyphene, chlophedianol, and fominoben, have poor affinity for DM sites whereas the antitussive noscapine enhances DM binding by increasing the affinity of DM for its central binding sites. Additional competition studies indicate that there is no correlation of DM binding with any of the known or putative neurotransmitters in the central nervous system. DM binding is also not related to tricyclic antidepressant binding sites or biogenic amine uptake sites. However, certain phenothiazine neuroleptics and typical and atypical antidepressants inhibit binding with IC50 values in the nanomolar range. Moreover, the anticonvulsant drug diphenylhydantoin enhances DM binding in a manner similar to that of noscapine. Preliminary experiments utilizing acid extracts of brain have not demonstrated the presence of an endogenous ligand for DM sites. The binding characteristics of DM sites studied in rat and mouse brain indicate that the relative potencies of several antitussives to inhibit specific DM binding vary according to species. High-affinity, saturable, and stereoselective [3H]DM binding sites are present in liver homogenates, but several differences have been found for these peripheral binding sites and those described for brain. Although the nature of central DM binding sites is not known, the potent interaction of several classes of centrally

  9. Probing conformational changes in lipoxygenases upon membrane binding: fine-tuning by the active site inhibitor ETYA.

    PubMed

    Di Venere, Almerinda; Nicolai, Eleonora; Ivanov, Igor; Dainese, Enrico; Adel, Susan; Angelucci, B C; Kuhn, Hartmut; Maccarrone, Mauro; Mei, Giampiero

    2014-01-01

    Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors. PMID:24012824

  10. A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics*

    PubMed Central

    McCoy, Jason G.; Rusinova, Radda; Kim, Dorothy M.; Kowal, Julia; Banerjee, Sourabh; Jaramillo Cartagena, Alexis; Thompson, Ameer N.; Kolmakova-Partensky, Ludmila; Stahlberg, Henning; Andersen, Olaf S.; Nimigean, Crina M.

    2014-01-01

    Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel. PMID:24515111

  11. MicroRNA binding sites in C. elegans 3' UTRs.

    PubMed

    Liu, Chaochun; Rennie, William A; Mallick, Bibekanand; Kanoria, Shaveta; Long, Dang; Wolenc, Adam; Carmack, C Steven; Ding, Ye

    2014-01-01

    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Since the discovery of lin-4, the founding member of the miRNA family, over 360 miRNAs have been identified for Caenorhabditis elegans (C. elegans). Prediction and validation of targets are essential for elucidation of regulatory functions of these miRNAs. For C. elegans, crosslinking immunoprecipitation (CLIP) has been successfully performed for the identification of target mRNA sequences bound by Argonaute protein ALG-1. In addition, reliable annotation of the 3' untranslated regions (3' UTRs) as well as developmental stage-specific expression profiles for both miRNAs and 3' UTR isoforms are available. By utilizing these data, we developed statistical models and bioinformatics tools for both transcriptome-scale and developmental stage-specific predictions of miRNA binding sites in C. elegans 3' UTRs. In performance evaluation via cross validation on the ALG-1 CLIP data, the models were found to offer major improvements over established algorithms for predicting both seed sites and seedless sites. In particular, our top-ranked predictions have a substantially higher true positive rate, suggesting a much higher likelihood of positive experimental validation. A gene ontology analysis of stage-specific predictions suggests that miRNAs are involved in dynamic regulation of biological functions during C. elegans development. In particular, miRNAs preferentially target genes related to development, cell cycle, trafficking, and cell signaling processes. A database for both transcriptome-scale and stage-specific predictions and software for implementing the prediction models are available through the Sfold web server at http://sfold.wadsworth.org. PMID:24827614

  12. Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

    PubMed

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward; Grallert, Harald; Glunk, Viktoria; Berulava, Tea; Lee, Heekyoung; Oskolkov, Nikolay; Fadista, Joao; Ehlers, Kerstin; Wahl, Simone; Hoffmann, Christoph; Qian, Kun; Rönn, Tina; Riess, Helene; Müller-Nurasyid, Martina; Bretschneider, Nancy; Schroeder, Timm; Skurk, Thomas; Horsthemke, Bernhard; Spieler, Derek; Klingenspor, Martin; Seifert, Martin; Kern, Michael J; Mejhert, Niklas; Dahlman, Ingrid; Hansson, Ola; Hauck, Stefanie M; Blüher, Matthias; Arner, Peter; Groop, Leif; Illig, Thomas; Suhre, Karsten; Hsu, Yi-Hsiang; Mellgren, Gunnar; Hauner, Hans; Laumen, Helmut

    2014-01-16

    Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms. PMID:24439387

  13. The Lipid Bilayer Provides a Site for Cortisone Crystallization at High Cortisone Concentrations.

    PubMed

    Alsop, Richard J; Khondker, Adree; Hub, Jochen S; Rheinstädter, Maikel C

    2016-01-01

    Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as "steroid flares" which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization. PMID:26936102

  14. The Lipid Bilayer Provides a Site for Cortisone Crystallization at High Cortisone Concentrations

    PubMed Central

    Alsop, Richard J.; Khondker, Adree; Hub, Jochen S.; Rheinstädter, Maikel C.

    2016-01-01

    Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as “steroid flares” which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization. PMID:26936102

  15. Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

    PubMed

    Liu, Yi; Chang, Sheng; Defersha, Fantahun M

    2015-07-01

    This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 < pKa < 9.0) were found to be readily removed by 0.45-μm membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling. PMID:25935368

  16. Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

    PubMed Central

    Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

    1999-01-01

    A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

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

    PubMed

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

    2001-12-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far. PMID:11722929

  18. Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

    PubMed Central

    Herrero, Salvador; González-Cabrera, Joel; Tabashnik, Bruce E.; Ferré, Juan

    2001-01-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far. PMID:11722929

  19. Turnover of binding sites for transcription factors involved in early Drosophila development.

    PubMed

    Costas, Javier; Casares, Fernando; Vieira, Jorge

    2003-05-22

    Despite the importance of cis-regulatory regions in evolution, little is know about their evolutionary dynamics. In this report, we analyze the process of evolution of binding sites for transcription factors using as a model a well characterized system, the Drosophila early developmental enhancers. We compare the sequences of eight enhancer regions for early developmental genes between Drosophila melanogaster and other two species, Drosophila virilis and Drosophila pseudoobscura, searching for the presence/absence of 104 biochemically verified binding sites from D. melanogaster. We also modeled the binding specificity of each binding site by the use of well-defined positional weight matrices (PWMs). The comparisons showed that turnover of binding sites seems to fit a molecular clock, at an approximate rate of 0.94% of gain/loss of binding sites per million years. This intense turnover affects both high and low affinity binding sites at the same extent. Furthermore, the subset of overlapping binding sites is also subjected to this high turnover. Conserved binding sites seem to be constrained to maintain not only location but also the exact sequence at each particular position. Finally, we detected a significant decrease in mean PWM scores for the D. virilis binding sites in the case of Hunchback. Possible explanations for this fact are discussed. PMID:12801649

  20. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

  1. CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

    PubMed

    Paes, Marcia C; Silveira, Alan B; Ventura-Martins, Guilherme; Luciano, Monalisa; Coelho, Marsen G P; Todeschini, Adriane R; Bianconi, M Lucia; Atella, Georgia C; Silva-Neto, Mário A C

    2015-10-01

    Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation. PMID:26111116

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

    PubMed

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

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

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

    PubMed Central

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

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  4. Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A.

    PubMed

    Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja; Janzen, Constantin; Lou, Jianlong; Marks, James D; Binz, Thomas; Rummel, Andreas

    2014-04-01

    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties. PMID:24583011

  5. Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS.

    PubMed Central

    Jarvis, B W; Lichenstein, H; Qureshi, N

    1997-01-01

    An early event in septic shock is the activation of macrophages by a complex consisting of lipopolysaccharide (LPS), LPS-binding protein (LBP), and the cell surface antigen CD14. The complexes that form between [3H]ReLPS (ReLPS is deep-rough-chemotype hexacyl LPS from E. coli D31m4), soluble CD14 (sCD14), and LBP were analyzed by two independent methods, native (nondenaturing) gel electrophoresis and size-exclusion high-performance liquid chromatography (HPLC). This is the first reported use of HPLC to purify and study LPS-protein complexes. The binding of [3H]ReLPS to LBP and sCD14 was inhibited by preincubation with diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA), a potent LPS antagonist. In addition, [3H]ReLPS bound to LBP and to a truncated form of sCD14 [sCD14(1-152)] that contained the LPS binding domain. Binding to both proteins was blocked by RsDPLA. Thus, RsDPLA competes in a 1:1 ratio for the same or nearby binding sites on ReLPS complexes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aggregated ReLPS eluting from the HPLC indicated that only LBP, not sCD14, was bound to the aggregated ReLPS. This finding supports the binary model of LPS complex formation with LBP and sCD14. PMID:9234747

  6. Lipid-based nanoparticles with high binding affinity for amyloid-beta1-42 peptide.

    PubMed

    Gobbi, Marco; Re, Francesca; Canovi, Mara; Beeg, Marten; Gregori, Maria; Sesana, Silvia; Sonnino, Sandro; Brogioli, Doriano; Musicanti, Claudia; Gasco, Paolo; Salmona, Mario; Masserini, Massimo E

    2010-09-01

    The neurotoxic beta-amyloid peptide (Abeta), formed in anomalous amounts in Alzheimer's disease (AD), is released as monomer and then undergoes aggregation forming oligomers, fibrils and plaques in diseased brains. Abeta aggregates are considered as possible targets for therapy and/or diagnosis of AD. Since nanoparticles (NPs) are promising vehicles for imaging probes and therapeutic agents, we realized and characterized two types of NPs (liposomes and solid lipid nanoparticles, 145 and 76 nm average size, respectively) functionalized to target Abeta(1-42) with high affinity. Preliminary immunostaining studies identified anionic phospholipids [phosphatidic acid (PA) and cardiolipin (CL)] as suitable Abeta(1-42) ligands. PA/CL-functionalized, but not plain, NPs interacted with Abeta(1-42) aggregates as indicated by ultracentrifugation experiments, in which binding reaction occurred in solution, and by Surface Plasmon Resonance (SPR) experiments, in which NPs flowed onto immobilized Abeta(1-42). All these experiments were carried out in buffered saline. SPR studies indicated that, when exposed on NPs surface, PA/CL display very high affinity for Abeta(1-42) fibrils (22-60 nm), likely because of the occurrence of multivalent interactions which markedly decrease the dissociation of PA/CL NPs from Abeta. Noteworthy, PA/CL NPs did not bind to bovine serum albumin. The PA/CL NPs described in this work are endowed with the highest affinity for Abeta so far reported. These characteristics make our NPs a very promising vector for the targeted delivery of potential new diagnostic and therapeutic molecules to be tested in appropriate animal models. PMID:20553982

  7. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  8. Lipid Selectivity, Orientation, and Extent of Membrane Binding of Nonacylated RP2.

    PubMed

    Demers, Éric; Boisselier, Élodie; Horchani, Habib; Blaudez, Daniel; Calvez, Philippe; Cantin, Line; Belley, Nicolas; Champagne, Sophie; Desbat, Bernard; Salesse, Christian

    2015-04-28

    Retinitis pigmentosa 2 (RP2) is an ubiquitary protein of 350 residues. The N-terminus of RP2 contains putative sites of myristoylation and palmitoylation. The dually acylated protein is predominantly localized to the plasma membrane. However, clinically occurring substitution mutations of RP2 in photoreceptors lead to the expression of a nonacylated protein, which was shown to be misrouted to intracellular organelles using different cell lines. However, the parameters responsible for the modulation of the membrane binding of nonacylated RP2 (naRP2) are still largely unknown. The maximal insertion pressure of naRP2 has thus been determined after its injection into the subphase underneath monolayers of phospholipids, which are typical of photoreceptor membranes. These data demonstrated that naRP2 shows a preferential binding to saturated phospholipid monolayers. Moreover, polarization modulation infrared reflection absorption spectroscopy has allowed comparison of the secondary structure of this protein in solution and upon binding to phospholipid monolayers. In addition, simulations of these spectra have allowed to determine that the β-helix of naRP2 has an orientation of 60° with respect to the normal, which remains unchanged regardless of the type of phospholipid. Finally, ellipsometric measurements of naRP2 demonstrated that its particular affinity for saturated phospholipids can be explained by its larger extent of insertion in this phospholipid monolayer compared to that in polyunsaturated phospholipid monolayers. PMID:25844643

  9. Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

    PubMed Central

    Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

    1991-01-01

    1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

  10. Vinculin acts as a sensor in lipid regulation of adhesion-site turnover.

    PubMed

    Chandrasekar, Indra; Stradal, Theresia E B; Holt, Mark R; Entschladen, Frank; Jockusch, Brigitte M; Ziegler, Wolfgang H

    2005-04-01

    The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP(2)-dependent disassembly of adhesions was suppressed. Thus, PIP(2) binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP(2) levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor. PMID:15769850

  11. Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

    SciTech Connect

    Montoudis, Alain; Delvin, Edgard; Menard, Daniel

    2006-01-06

    Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.

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

    PubMed Central

    2009-01-01

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

  13. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    SciTech Connect

    Sigalov, Alexander B.; Hendricks, Gregory M.

    2009-11-13

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including {zeta}{sub cyt} and CD3{epsilon}{sub cyt} all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  14. Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7.

    PubMed

    Zahn, Michael; Kieslich, Bjorn; Berthold, Nicole; Knappe, Daniel; Hoffmann, Ralf; Strater, Norbert

    2014-04-01

    Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified. PMID:24164259

  15. COTRASIF: conservation-aided transcription-factor-binding site finder.

    PubMed

    Tokovenko, Bogdan; Golda, Rostyslav; Protas, Oleksiy; Obolenskaya, Maria; El'skaya, Anna

    2009-04-01

    COTRASIF is a web-based tool for the genome-wide search of evolutionary conserved regulatory regions (transcription factor-binding sites, TFBS) in eukaryotic gene promoters. Predictions are made using either a position-weight matrix search method, or a hidden Markov model search method, depending on the availability of the matrix and actual sequences of the target TFBS. COTRASIF is a fully integrated solution incorporating both a gene promoter database (based on the regular Ensembl genome annotation releases) and both JASPAR and TRANSFAC databases of TFBS matrices. To decrease the false-positives rate an integrated evolutionary conservation filter is available, which allows the selection of only those of the predicted TFBS that are present in the promoters of the related species' orthologous genes. COTRASIF is very easy to use, implements a regularly updated database of promoters and is a powerful solution for genome-wide TFBS searching. COTRASIF is freely available at http://biomed.org.ua/COTRASIF/. PMID:19264796

  16. Every Site Counts: Submitting Transcription Factor-Binding Site Information through the CollecTF Portal.

    PubMed

    Erill, Ivan

    2015-08-01

    Experimentally verified transcription factor-binding sites represent an information-rich and highly applicable data type that aptly summarizes the results of time-consuming experiments and inference processes. Currently, there is no centralized repository for this type of data, which is routinely embedded in articles and extremely hard to mine. CollecTF provides the first standardized resource for submission and deposition of these data into the NCBI RefSeq database, maximizing its accessibility and prompting the community to adopt direct submission policies. PMID:26013488

  17. Calcium binding to human platelet integrin GPIIb/IIIa and to its constituent glycoproteins. Effects of lipids and temperature.

    PubMed Central

    Rivas, G A; González-Rodríguez, J

    1991-01-01

    Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer. GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca(2-)-binding sites (Kd0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb alpha-(1-285) and GPIIb alpha-(314-489), which carry three out of the four putative Ca(2+)-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca(2+)-binding site (Kd1 80 +/- 30 nM at 21 degrees C), whose degree of saturation regulates the state of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca(2+)-binding sites (Kd2 40 +/- 15 microM at 21 degrees C). When GPIIb/IIIa was incorporated into liposomes, Kd1 decreased by an order of magnitude (9 +/- 3 nM at 21 degrees C) and reached the dissociation constant estimated for the high-affinity Ca(2+)-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas Kd2 remained unchanged. The high-affinity Ca(2+)-binding site of GPIIb/IIIa in solution at 4 degrees C has almost the same affinity (Kd1 65 +/- 20 nM) as at 21 degrees C; however, at 37 degrees C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca(2+)-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, Kd 25 +/- 11 microM), or vanishes altogether. Studies on Ca(2+)-dependent dissociation of GPIIIb/IIIa at 37 degrees C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane

  18. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    SciTech Connect

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  19. Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

    PubMed Central

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

    2015-01-01

    The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

  20. Characterization of the Estradiol-Binding Site Structure of Human Protein Disulfide Isomerase (PDI)

    PubMed Central

    Fu, Xin-Miao; Wang, Pan; Zhu, Bao Ting

    2011-01-01

    Background Earlier studies showed that 17β-estradiol (E2), an endogenous female sex hormone, can bind to human protein disulfide isomerase (PDI), a protein folding catalyst for disulfide bond formation and rearrangement. This binding interaction can modulate the intracellular levels of E2 and its biological actions. However, the structure of PDI's E2-binding site is still unclear at present, which is the focus of this study. Methodology/Principal Findings The E2-binding site structure of human PDI was studied by using various biochemical approaches coupled with radiometric receptor-binding assays, site-directed mutagenesis, and molecular computational modeling. Analysis of various PDI protein fragments showed that the [3H]E2-binding activity is not associated with the single b or b' domain but is associated with the b-b' domain combination. Computational docking analyses predicted that the E2-binding site is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. A hydrogen bond, formed between the 3-hydroxyl group of E2 and His256 of PDI is critical for the binding interaction. This binding model was jointly confirmed by a series of detailed experiments, including site-directed mutagenesis of the His256 residue coupled with selective modifications of the ligand structures to alter the binding interaction. Conclusions/Significance The results of this study elucidated the structural basis for the PDI–E2 binding interaction and the reservoir role of PDI in modulating the intracellular E2 levels. The identified PDI E2-binding site is quite different from its known peptide binding sites. Given that PDI is a potential therapeutic target for cancer chemotherapy and HIV prevention and that E2 can inhibit PDI activity in vitro, the E2-binding site structure of human PDI determined here offers structural insights which may aid in the rational design of novel PDI inhibitors. PMID:22073283

  1. Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion.

    PubMed

    De Haro, Luc; Quetglas, Stéphanie; Iborra, Cécile; Lévêque, Christian; Seagar, Michael

    2003-10-01

    Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane. PMID:14597264

  2. Duplicate gene divergence by changes in microRNA binding sites in Arabidopsis and Brassica.

    PubMed

    Wang, Sishuo; Adams, Keith L

    2015-03-01

    Gene duplication provides large numbers of new genes that can lead to the evolution of new functions. Duplicated genes can diverge by changes in sequences, expression patterns, and functions. MicroRNAs play an important role in the regulation of gene expression in many eukaryotes. After duplication, two paralogs may diverge in their microRNA binding sites, which might impact their expression and function. Little is known about conservation and divergence of microRNA binding sites in duplicated genes in plants. We analyzed microRNA binding sites in duplicated genes in Arabidopsis thaliana and Brassica rapa. We found that duplicates are more often targeted by microRNAs than singletons. The vast majority of duplicated genes in A. thaliana with microRNA binding sites show divergence in those sites between paralogs. Analysis of microRNA binding sites in genes derived from the ancient whole-genome triplication in B. rapa also revealed extensive divergence. Paralog pairs with divergent microRNA binding sites show more divergence in expression patterns compared with paralog pairs with the same microRNA binding sites in Arabidopsis. Close to half of the cases of binding site divergence are caused by microRNAs that are specific to the Arabidopsis genus, indicating evolutionarily recent gain of binding sites after target gene duplication. We also show rapid evolution of microRNA binding sites in a jacalin gene family. Our analyses reveal a dynamic process of changes in microRNA binding sites after gene duplication in Arabidopsis and highlight the role of microRNA regulation in the divergence and contrasting evolutionary fates of duplicated genes. PMID:25644246

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

    SciTech Connect

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

    2006-04-19

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

  4. Molecular dynamics simulations of barley and maize lipid transfer proteins show different ligand binding preferences in agreement with experimental data.

    PubMed

    Smith, Lorna J; Roby, Ysobel; Allison, Jane R; van Gunsteren, Wilfred F

    2013-07-30

    Experimental studies of barley and maize lipid transfer proteins (LTPs) show that the two proteins bind the ligand palmitate in opposite orientations in their internal cavities. Moreover, maize LTP is reported to bind the ligand caprate in the internal cavity in a mixture of two orientations with approximately equal occupancy. Six 30 ns molecular dynamics (MD) simulations of maize and barley LTP with ligands bound in two orientations (modes M and B) have been used to understand the different ligand binding preferences. The simulations show that both maize and barley LTP could bind palmitate in the orientation observed experimentally for maize LTP (mode M), with the predominant interaction being a salt bridge between the ligand carboxylate headgroup and a conserved arginine side chain. However, the simulation of barley LTP with palmitate in the mode B orientation shows the most favorable protein-ligand interaction energy. In contrast, the simulations of maize LTP with palmitate and with caprate in the mode B orientation show no persistent ligand binding, the ligands leaving the cavity during the simulations. Sequence differences between maize and barley LTP in the AB loop region, in residues at the base of the hydrophobic cavity, and in the helix A region are identified as contributing to the different behavior. The simulations reproduce well the experimentally observed binding preferences for palmitate and suggest that the experimental data for maize LTP with caprate reflect ligand mobility in binding mode M rather than the population of binding modes M and B. PMID:23834513

  5. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    SciTech Connect

    James, I.F.; Goldstein, A.

    1984-05-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, (/sup 3/H) dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for (/sup 3/H) (D-Ala2, D-Leu5)enkephalin and (3H)ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites.

  6. Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy

    PubMed Central

    Banerjee, Amrita; Perez-Castillejos, R.; Hahn, D.; Smirnov, Alex I.; Grebel, H.

    2013-01-01

    We used Surface Enhanced Raman Spectroscopy (SERS) to detect binding events between streptavidin and biotinylated lipid bilayers. The binding events took place at the surface between microfluidic channels and anodized aluminum oxide (AAO) with the latter serving as substrates. The bilayers were incorporated in the substrate pores. It was revealed that non-bound molecules were easily washed away and that large suspended cells (Salmonella enterica) are less likely to interfere with the monitoring process: when focusing to the lower surface of the channel, one may resolve mostly the bound molecules. PMID:24932024

  7. Lipid oxidation in trout muscle is strongly inhibited by a protein that specifically binds hemin released from hemoglobin

    PubMed Central

    Cai, He; Grunwald, Eric W; Park, Sungyong; Lei, Benfang; Richards, Mark P.

    2013-01-01

    The recombinant Streptococcal protein apoShp can be used as a probe for hemoglobin (Hb) reactivity in fish muscle due to its specific affinity for hemin that is released from Hb at post mortem pH values. Hemin affinity measurements indicated that apoShp binds hemin released from Hb but not myoglobin (Mb). Hemin affinity of holoShp was higher at pH 5.7 compared to pH 8.0. This may be attributed to enhanced electrostatic interaction of His58 with the heme-7-propionate at lower pH. ApoShp readily acquired hemin that was released from trout IV metHb in the presence of washed cod muscle during 2°C storage at pH 6.3. This was based on increases in redness in the washed cod matrix which occurs when apoShp binds hemin that is released from metHb. ApoShp prevented Hb-mediated lipid oxidation in washed cod muscle during 2°C storage. The prevention of Hb-mediated lipid oxidation by apoShp was likely due to bis-methionyl coordination of hemin that dissociated from metHb. This hexa-coordination of hemin appears to prevent peroxide-mediated redox reactions and there is no component in the matrix capable of dissociating hemin from Shp. ApoShp was also added to minced muscle from Rainbow trout (Oncorhynchus mykiss) to examine the degree to which Hb contributes to lipid oxidation in trout muscle. Addition of apoShp inhibited approximately 90% of the lipid oxidation that occurred in minced trout muscle during 9 days of 2°C storage based on lipid peroxide, hexanal, and thiobarituric acid reactive substances (TBARS) values. These results strongly suggest that Hb is the primary promoter of lipid oxidation in trout muscle. PMID:23570608

  8. Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

    PubMed

    Adams, E M; Lubrano, T M; Gordon, J; Fields, J Z

    1992-09-01

    We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor. PMID:1392105

  9. Functional Linkage of Adenine Nucleotide Binding Sites in Mammalian Muscle 6-Phosphofructokinase*

    PubMed Central

    Brüser, Antje; Kirchberger, Jürgen; Kloos, Marco; Sträter, Norbert; Schöneberg, Torsten

    2012-01-01

    6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution). PMID:22474333

  10. Membrane Thinning Due to Antimicrobial Peptide Binding: An Atomic Force Microscopy Study of MSI-78 in Lipid Bilayers

    PubMed Central

    Mecke, Almut; Lee, Dong-Kuk; Ramamoorthy, Ayyalusamy; Orr, Bradford G.; Banaszak Holl, Mark M.

    2005-01-01

    The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 ± 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly. PMID:16183881

  11. Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

    PubMed Central

    Ioannou, Charikleia; Schaeffer, Patrick M.; Dixon, Nicholas E.; Soultanas, Panos

    2006-01-01

    The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular ‘switch’ regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system. PMID:17003052

  12. Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis.

    PubMed

    Ioannou, Charikleia; Schaeffer, Patrick M; Dixon, Nicholas E; Soultanas, Panos

    2006-01-01

    The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system. PMID:17003052

  13. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    PubMed Central

    Lin, Yu-Feng; Chen, Jin-Yi

    2015-01-01

    We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified. PMID:26000290

  14. Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila

    PubMed Central

    Adryan, Boris; Woerfel, Gertrud; Birch-Machin, Ian; Gao, Shan; Quick, Marie; Meadows, Lisa; Russell, Steven; White, Robert

    2007-01-01

    Background Insulator elements are proposed to play a key role in the organization of the regulatory architecture of the genome. In Drosophila, one of the best studied is the gypsy retrotransposon insulator, which is bound by the Suppressor of Hairy-wing (Su [Hw]) transcriptional regulator. Immunolocalization studies suggest that there are several hundred Su(Hw) sites in the genome, but few of these endogenous Su(Hw) binding sites have been identified. Results We used chromatin immunopurification with genomic microarray analysis to identify in vivo Su(Hw) binding sites across the 3 megabase Adh region. We find 60 sites, and these enabled the construction of a robust new Su(Hw) binding site consensus. In contrast to the gypsy insulator, which contains tightly clustered Su(Hw) binding sites, endogenous sites generally occur as isolated sites. These endogenous sites have three key features. In contrast to most analyses of DNA-binding protein specificity, we find that strong matches to the binding consensus are good predictors of binding site occupancy. Examination of occupancy in different tissues and developmental stages reveals that most Su(Hw) sites, if not all, are constitutively occupied, and these isolated Su(Hw) sites are generally highly conserved. Analysis of transcript levels in su(Hw) mutants indicate widespread and general changes in gene expression. Importantly, the vast majority of genes with altered expression are not associated with clustering of Su(Hw) binding sites, emphasizing the functional relevance of isolated sites. Conclusion Taken together, our in vivo binding and gene expression data support a role for the Su(Hw) protein in maintaining a constant genomic architecture. PMID:17705839

  15. Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

    PubMed Central

    Schubert, David A.; Gordo, Susana; Chu, H. Hamlet

    2012-01-01

    The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation. PMID:23166358

  16. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  17. Active site and laminarin binding in glycoside hydrolase family 55.

    PubMed

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

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

    PubMed

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

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. PMID:26896718

  19. Using circular permutation analysis to redefine the R17 coat protein binding site.

    PubMed

    Gott, J M; Pan, T; LeCuyer, K A; Uhlenbeck, O C

    1993-12-14

    The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used. PMID:7504949

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

    SciTech Connect

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

    1988-01-01

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

  1. Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

    SciTech Connect

    Muccioli, G.; Guardabassi, A.; Pattono, P. )

    1990-03-01

    The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrence of prolactin specific binding sites in Xenopus laevis choroid plexus cells.

  2. 2-([sup 125]I) iodomelatonin binding sites in rat adrenals: Pharmacological characteristics and subcellular distribution

    SciTech Connect

    Persengiev, S.P. )

    1992-01-01

    Specific binding sites for 2-[[sup 125]I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[[sup 125]I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin > 2-iodomelatonin > melatonin > 5-methoxytryptamine > 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear and mitochondrial subcellular fractions.

  3. ABC transporters: one, two or four extracytoplasmic substrate-binding sites?

    PubMed Central

    van der Heide, Tiemen; Poolman, Bert

    2002-01-01

    Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator. PMID:12370206

  4. Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland.

    PubMed

    Beaujouan, J C; Saffroy, M; Torrens, Y; Sagan, S; Glowinski, J

    1999-11-01

    Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue. PMID:10612450

  5. Identification of two uridine binding domain peptides of the UDP-glucose-binding site of rabbit muscle glycogenin.

    PubMed

    Carrizo, M E; Curtino, J A

    1998-12-30

    Glycogenin, the autoglucosyltransferase that initiates the de novo biosynthesis of glycogen, photoaffinity labeled with [beta32P]5-azido-UDP-glucose. The photoinsertion of the azidouridine derivative showed activating ultraviolet light dependency, saturation effects, and inhibition by UDP-glucose, thus demonstrating the specificity of the interaction. In the absence of Mn2+, the requirement for the catalytic activity of glycogenin, the photolabeling decreased by 70%. Competitive binding experiments indicated that the pyrophosphate or a phosphate was the moiety of UDP-glucose implicated in the strongest interaction at the binding site. Proteolytic digestion of photolabeled glycogenin resulted in the identification of two labeled fragments, 89-143 and 168-233, that carried the uridine binding sites. This is the first report of the region of glycogenin that harbors the UDP-glucose-binding domain. PMID:9918805

  6. Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers.

    PubMed

    Miotto, Benoit; Ji, Zhe; Struhl, Kevin

    2016-08-16

    The origin recognition complex (ORC) binds sites from which DNA replication is initiated. We address ORC binding selectivity in vivo by mapping ∼52,000 ORC2 binding sites throughout the human genome. The ORC binding profile is broader than those of sequence-specific transcription factors, suggesting that ORC is not bound or recruited to specific DNA sequences. Instead, ORC binds nonspecifically to open (DNase I-hypersensitive) regions containing active chromatin marks such as H3 acetylation and H3K4 methylation. ORC sites in early and late replicating regions have similar properties, but there are far more ORC sites in early replicating regions. This suggests that replication timing is due primarily to ORC density and stochastic firing of origins. Computational simulation of stochastic firing from identified ORC sites is in accord with replication timing data. Large genomic regions with a paucity of ORC sites are strongly associated with common fragile sites and recurrent deletions in cancers. We suggest that replication origins, replication timing, and replication-dependent chromosome breaks are determined primarily by the genomic distribution of activator proteins at enhancers and promoters. These activators recruit nucleosome-modifying complexes to create the appropriate chromatin structure that allows ORC binding and subsequent origin firing. PMID:27436900

  7. Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

    PubMed Central

    Smith, Lorna J; Gunsteren, Wilfred F Van; Allison, Jane R

    2013-01-01

    Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force-field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen-bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints. PMID:23139016

  8. Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    PubMed Central

    2013-01-01

    Background Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. Results We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction. Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1

  9. Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis.

    PubMed

    Trexler, Adam J; Sochacki, Kem A; Taraska, Justin W

    2016-08-01

    How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells. PMID:27307587

  10. Probing the Binding Site of Bile Acids in TGR5.

    PubMed

    Macchiarulo, Antonio; Gioiello, Antimo; Thomas, Charles; Pols, Thijs W H; Nuti, Roberto; Ferrari, Cristina; Giacchè, Nicola; De Franco, Francesca; Pruzanski, Mark; Auwerx, Johan; Schoonjans, Kristina; Pellicciari, Roberto

    2013-12-12

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  11. Comparing binding site information to binding affinity reveals that Crp/DNA complexes have several distinct binding conformers

    PubMed Central

    Holmquist, Peter C.; Holmquist, Gerald P.; Summers, Michael L.

    2011-01-01

    We show that the cAMP receptor protein (Crp) binds to DNA as several different conformers. This situation has precluded discovering a high correlation between any sequence property and binding affinity for proteins that bend DNA. Experimentally quantified affinities of Synechocystis sp. PCC 6803 cAMP receptor protein (SyCrp1), the Escherichia coli Crp (EcCrp, also CAP) and DNA were analyzed to mathematically describe, and make human-readable, the relationship of DNA sequence and binding affinity in a given system. Here, sequence logos and weight matrices were built to model SyCrp1 binding sequences. Comparing the weight matrix model to binding affinity revealed several distinct binding conformations. These Crp/DNA conformations were asymmetrical (non-palindromic). PMID:21586590

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

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

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

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  14. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  15. Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding Mechanism on Lipid Bilayers Studied by QCM-D and AFM.

    PubMed

    Tharad, Sudarat; Iturri, Jagoba; Moreno-Cencerrado, Alberto; Mittendorfer, Margareta; Promdonkoy, Boonhiang; Krittanai, Chartchai; Toca-Herrera, José L

    2015-09-29

    Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, including the cytolytic protein (Cyt2Aa2), which is a bioinsecticide and an antimicrobial protein. However, the binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers is still unclear. In this work, we have used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers. We have found that the binding mechanism is concentration dependent. While at 10 μg/mL, Cyt2Aa2 binds slowly on the lipid bilayer forming a compliance protein/lipid layer with aggregates, at higher protein concentrations (100 μg/mL), the binding is fast, and the protein/lipid layer is more rigid including holes (of about a lipid bilayer thickness) in its structure. Our study suggests that the protein/lipid bilayer binding mechanism seems to be carpet-like at low protein concentrations and pore forming-like at high protein concentrations. PMID:26354323

  16. Detecting O2 binding sites in protein cavities

    PubMed Central

    Kitahara, Ryo; Yoshimura, Yuichi; Xue, Mengjun; Kameda, Tomoshi; Mulder, Frans A. A.

    2016-01-01

    Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins. PMID:26830762

  17. Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer.

    PubMed

    Fox, Jamie C; Tyler, Robert C; Peterson, Francis C; Dyer, Douglas P; Zhang, Fuming; Linhardt, Robert J; Handel, Tracy M; Volkman, Brian F

    2016-03-01

    Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition. PMID:26836755

  18. Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I*

    PubMed Central

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

    2013-01-01

    Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-NG and ω-NG′ nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant. PMID:23836892

  19. A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors.

    PubMed

    Saleh, Noureldin; Saladino, Giorgio; Gervasio, Francesco L; Haensele, Elke; Banting, Lee; Whitley, David C; Sopkova-de Oliveira Santos, Jana; Bureau, Ronan; Clark, Timothy

    2016-07-01

    Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity. PMID:27184628

  20. Low affinity binding site clusters confer hox specificity and regulatory robustness.

    PubMed

    Crocker, Justin; Abe, Namiko; Rinaldi, Lucrezia; McGregor, Alistair P; Frankel, Nicolás; Wang, Shu; Alsawadi, Ahmad; Valenti, Philippe; Plaza, Serge; Payre, François; Mann, Richard S; Stern, David L

    2015-01-15

    In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression. PMID:25557079

  1. Phospholipid-binding sites of phosphatase and tensin homolog (PTEN): exploring the mechanism of phosphatidylinositol 4,5-bisphosphate activation.

    PubMed

    Wei, Yang; Stec, Boguslaw; Redfield, Alfred G; Weerapana, Eranthie; Roberts, Mary F

    2015-01-16

    The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide (31)P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling (31)P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis. PMID:25429968

  2. 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

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

    SciTech Connect

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

    2003-08-28

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

  4. 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

  5. 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

  6. Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

    PubMed Central

    Águila, Sonia; Teruel-Montoya, Raúl; Vicente, Vicente; Corral, Javier; Martínez-Martínez, Irene

    2016-01-01

    Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin. PMID:27322195

  7. Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

    SciTech Connect

    Itzhak, Y.

    1988-01-01

    The pharmacological specificity of representative psychotomimetic agents such a phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with (/sup 3/H)-1-(1-(3-hydroxyphenyl) cyclohexyl) piperidine ((/sup 3/H)PCP-3-OH), (+)(/sup 3/H)-N-allylnormetazocine ((+)(/sup 3/H)SKF 10047) and (+) (/sup 3/H)-3-(3-hydroxy-phenyl)-N-(1-propyl) piperidine and ((+)(/sup 3/H)-3-PPP). PCP analogs inhibit potently high affinity (/sup 3/H)PCP-3-OH binding and (+)(/sup 3/H)SKF 10047 binding, moderately the low affinity binding component of (/sup 3/H)PCP-3-OH and very weakly (+) (/sup 3/H)-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+)(/sup 3/H)SKF 10047, high affinity (/sup 3/H)PCP-3-OH and (+)(/sup 3/H)-3-PCP-3-OH binding. The antipsychotic agents display high affinity for (+)(/sup 3/H)-3-PPP binding sites, moderate affinity for (+)(/sup 3/H)SKF 10047 sites and have no effect on either the high or low affinity (/sup 3/H)PCP-3-OH binding. 20 references, 3 figures, 2 tables.

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

    SciTech Connect

    Crankshaw, D.; Gaspar, V.; Pliska, V. )

    1990-01-01

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

  9. Phosfinder: a web server for the identification of phosphate-binding sites on protein structures.

    PubMed

    Parca, Luca; Mangone, Iolanda; Gherardini, Pier Federico; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2011-07-01

    Phosfinder is a web server for the identification of phosphate binding sites in protein structures. Phosfinder uses a structural comparison algorithm to scan a query structure against a set of known 3D phosphate binding motifs. Whenever a structural similarity between the query protein and a phosphate binding motif is detected, the phosphate bound by the known motif is added to the protein structure thus representing a putative phosphate binding site. Predicted binding sites are then evaluated according to (i) their position with respect to the query protein solvent-excluded surface and (ii) the conservation of the binding residues in the protein family. The server accepts as input either the PDB code of the protein to be analyzed or a user-submitted structure in PDB format. All the search parameters are user modifiable. Phosfinder outputs a list of predicted binding sites with detailed information about their structural similarity with known phosphate binding motifs, and the conservation of the residues involved. A graphical applet allows the user to visualize the predicted binding sites on the query protein structure. The results on a set of 52 apo/holo structure pairs show that the performance of our method is largely unaffected by ligand-induced conformational changes. Phosfinder is available at http://phosfinder.bio.uniroma2.it. PMID:21622655

  10. 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

  11. Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

    PubMed

    Zhuang, X; Wang, Q; Wang, B; Zhong, T; Cao, Y; Li, K; Yin, J

    2014-06-01

    The scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a predominant underground pest in the northern parts of China, and its larvae (grubs) cause great economic losses because of its wide range of host plants and covert habitats. Environmentally friendly strategies for controlling adults would have novel and broad potential applications. One potential pest management measure is the regulation of olfactory chemoreception to control target insect pests. In the process of olfactory recognition, odorant-binding proteins (OBPs) are believed to carry hydrophobic odorants from the environment to the surface of olfactory receptor neurons. To obtain a better understanding of the relationship between OBP structures and their ligands, homology modelling and molecular docking have been conducted on the interaction between HoblOBP1 and hexyl benzoate in the present study. Based on the results, site-directed mutagenesis and binding experiments were combined to describe the binding sites of HoblOBP1 and to explore its ligand-binding mechanism. After homology modelling of HoblOBP1, it was found that the three-dimensional structure of HoblOBP1 consists of six α-helices and three disulphide bridges that connect the helices, and the hydrophobic pockets are both composed of five helices. Based on the docking study, we found that van der Waals interactions and hydrophobic interactions are both important in the bonding between HoblOBP1 and hexyl benzoate. Intramolecular residues formed the hydrogen bonds in the C terminus of the protein and the bonds are crucial for the ligand-binding specificity. Finally, MET48, ILE80 and TYR111 are binding sites predicted for HoblOBP1. Using site-directed mutagenesis and fluorescence assays, it was found that ligands could not be recognized by mutant of Tyr111. A possible explanation is that the compound could not be recognized by the mutant, and remains in the binding cavity because of the loss of the intramolecular

  12. Locating the binding sites of antitumor drug tamoxifen and its metabolites with DNA.

    PubMed

    Bourassa, P; Thomas, T J; Tajmir-Riahi, H A

    2014-07-01

    We located the binding sites of antitumor drugs tamoxifen, 4-hydroxytamoxifen and endoxifen with calf-thymus DNA. FTIR, CD, UV-vis and fluorescence spectroscopic methods as well as molecular modeling were used to characterize the drug binding sites, binding constant and the effect of drug binding on DNA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bind DNA via hydrophobic and hydrophilic interactions with overall binding constants of K(tam-DNA)=3.5 (±0.2)×10⁴ M⁻¹, K(4-hydroxytam-DNA)=3.3 (±0.4) × 10⁴ M⁻¹ and K(endox)-DNA=2.8 (±0.8)×10⁴ M⁻¹. The number of binding sites occupied by drug is 1 (tamoxifen), 0.8 (4-hydroxitamoxifen) and 1.2 (endoxifen). Docking showed the participation of several nucleobases in drug-DNA complexes with the free binding energy of -3.85 (tamoxifen), -4.18 (4-hydroxtamoxifen) and -3.74 kcal/mol (endoxifen). The order of binding is 4-hydroxy-tamoxen>tamoxifen>endoxifen. Drug binding did not alter DNA conformation from B-family structure, while major biopolymer aggregation occurred at high drug concentrations. The drug binding mode is correlated with the mechanism of action of antitumor activity of tamoxifen and its metabolites. PMID:24682017

  13. Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

    SciTech Connect

    Leslie, R.A.; McDonald, T.J.; Robertson, H.A.

    1988-09-01

    Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared (/sup 125/I)PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of (/sup 125/I)PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of (/sup 125/I)PYY binding sites throughout the rat brain was seen to be similar to the distribution of (/sup 125/I)NPY binding sites.

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

    PubMed

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods. PMID:27046190

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

    PubMed

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

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. PMID:27318092

  16. Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

    PubMed Central

    Groenen, M A; Dijkhof, R J; van der Poel, J J; van Diggelen, R; Verstege, E

    1992-01-01

    Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. Images PMID:1508722

  17. High Membrane Curvature Enhances Binding, Conformational Changes, and Fibrillation of Amyloid-β on Lipid Bilayer Surfaces.

    PubMed

    Sugiura, Yuuki; Ikeda, Keisuke; Nakano, Minoru

    2015-10-27

    Aggregation of the amyloid-β (Aβ) protein and the formation of toxic aggregates are the possible pathogenic pathways in Alzheimer's disease. Accumulating evidence suggests that lipid membranes play key roles in protein aggregation, although the intermolecular forces that drive the interactions between Aβ-(1-40) and the membranes vary in different membrane systems. Here, we observed that a high positive curvature of lipid vesicles with diameters of ∼30 nm enhanced the association of Aβ with anionic phosphatidylglycerol membranes in the liquid-crystalline phase and with zwitterionic phosphatidylcholine membranes in the gel phase. The binding modes of Aβ to these membranes differ in terms of the location of the protein on the membrane and of the protein secondary structure. The fibrillation of Aβ was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein accumulated on the surfaces, as demonstrated by a high (10(7) M(-1)) binding constant. Our findings suggest that packing defects on membranes with high curvatures, such as the intraluminal vesicles in multivesicular bodies and the exosomes, might result in the accumulation of toxic protein aggregates. PMID:26474149

  18. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    PubMed

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  19. Evidence for two distinct binding sites for tau on microtubules

    PubMed Central

    Makrides, Victoria; Massie, Michelle R.; Feinstein, Stuart C.; Lew, John

    2004-01-01

    The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low- to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the tau-microtubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation/association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance. PMID:15096589

  20. Binding of the Cationic Peptide (KL)4K to Lipid Monolayers at the Air-Water Interface: Effect of Lipid Headgroup Charge, Acyl Chain Length, and Acyl Chain Saturation.

    PubMed

    Hädicke, André; Blume, Alfred

    2016-04-28

    The binding of the cationic peptide (KL)4K to monolayers of different anionic lipids was determined by adsorption experiments. The chemical structure of the anionic phospholipids was changed in different ways. First, the hydrophobic region of phosphatidylglycerols was altered by elongation of the acyl chain length. Second, an unsaturated chain was introduced. Third, lipids with negatively charged headgroups of different chemical structure were compared. (KL)4K itself shows no surface activity and does not bind to monolayers of zwitterionic lipids. Analysis of (KL)4K binding to anionic lipid monolayers reveals a competition between two binding processes: (i) incorporation of the peptide into the acyl chain region (surface pressure increase) and (ii) electrostatic interaction screening the negative charges with reduction of charge repulsion (surface pressure decrease due to monolayer condensation). The lipid acyl chain length and the chemical structure of the headgroup have minor effects on the binding properties. However, a strong dependence on the phase state of the monolayer was observed. In the liquid-expanded (LE) phase, the fluid monolayer provides enough space, so that peptide insertion due to hydrophobic interactions dominates. For monolayers in the liquid-condensed (LC) phase, peptide binding followed by monolayer condensation is the main effect. PMID:27049846

  1. Binding sites for L-(/sup 3/H)glutamate in hippocampus

    SciTech Connect

    Werling, L.L.

    1983-01-01

    Three binding sites for L-(/sup 3/H)glutamate on freshly-prepared hippocampal synaptic membranes were identified on the basis of their differing affinities for L-glutamate or quisqualate. The high affinity site yielded K/sub D/ and B/sub max/ values of 12 nM and 2.5 pmol/mg protein, respectively. Binding sites of lower affinity had K/sub D/ values of 200 nM (GLU A) and 1 ..mu..M (GLU B) and B/sub max/ values of about 30 and 60 pmol/mg protein, respectively. GLU A sites bound quisqualate with about 70 times the affinity fo GLU B sites, and thus quisoqualate could be used as a tool to discriminate them. Hill slopes indicated that each site represented a single population of non-interacting binding sites. Freezing drastically decreased GLU A binding, but nearly tripled GLU B binding. Both sites bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-asparate and discriminated poorly between L- and D-asparate. In contrast, the GLU B site bound L-aspartate with similar affinity to L-gluamate, and with much higher affinity than it bound D-aspartate. Both lesions of perforant path and destruction of the granule cells with colchicine markedly reduced radioligand binding to the GLU A site in the fascia dentata, but only the perforant path lesion significantly reduced binding to the GLU B site. The structural specificity of the GLU A site is consistent with its identification as a type of quisqualate receptor.

  2. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites.

    PubMed

    Lelieveld, Stefan H; Schütte, Judith; Dijkstra, Maurits J J; Bawono, Punto; Kinston, Sarah J; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-05-01

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. PMID:26721389

  3. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites

    PubMed Central

    Lelieveld, Stefan H.; Schütte, Judith; Dijkstra, Maurits J.J.; Bawono, Punto; Kinston, Sarah J.; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-01-01

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. PMID:26721389

  4. DISPLAR: an accurate method for predicting DNA-binding sites on protein surfaces

    PubMed Central

    Tjong, Harianto; Zhou, Huan-Xiang

    2007-01-01

    Structural and physical properties of DNA provide important constraints on the binding sites formed on surfaces of DNA-targeting proteins. Characteristics of such binding sites may form the basis for predicting DNA-binding sites from the structures of proteins alone. Such an approach has been successfully developed for predicting protein–protein interface. Here this approach is adapted for predicting DNA-binding sites. We used a representative set of 264 protein–DNA complexes from the Protein Data Bank to analyze characteristics and to train and test a neural network predictor of DNA-binding sites. The input to the predictor consisted of PSI-blast sequence profiles and solvent accessibilities of each surface residue and 14 of its closest neighboring residues. Predicted DNA-contacting residues cover 60% of actual DNA-contacting residues and have an accuracy of 76%. This method significantly outperforms previous attempts of DNA-binding site predictions. Its application to the prion protein yielded a DNA-binding site that is consistent with recent NMR chemical shift perturbation data, suggesting that it can complement experimental techniques in characterizing protein–DNA interfaces. PMID:17284455

  5. Host-Guest Binding-Site-Tunable Self-Assembly of Stimuli-Responsive Supramolecular Polymers.

    PubMed

    Yao, Hao; Qi, Miao; Liu, Yuyang; Tian, Wei

    2016-06-13

    Despite the remarkable progress made in controllable self-assembly of stimuli-responsive supramolecular polymers (SSPs), a basic issue that has not been consideration to date is the essential binding site. The noncovalent binding sites, which connect the building blocks and endow supramolecular polymers with their ability to respond to stimuli, are expected to strongly affect the self-assembly of SSPs. Herein, the design and synthesis of a dual-stimuli thermo- and photoresponsive Y-shaped supramolecular polymer (SSP2) with two adjacent β-cyclodextrin/azobenzene (β-CD/Azo) binding sites, and another SSP (SSP1) with similar building blocks, but only one β-CD/Azo binding site as a control, are described. Upon gradually increasing the polymer solution temperature or irradiating with UV light, SSP2 self-assemblies with a higher binding-site distribution density; exhibits a flower-like morphology, smaller size, and more stable dynamic aggregation process; and greater controllability for drug-release behavior than those observed with SSP1 self-assemblies. The host-guest binding-site-tunable self-assembly was attributed to the positive cooperativity generated among adjacent binding sites on the surfaces of SSP2 self-assemblies. This work is beneficial for precisely controlling the structural parameters and controlled release function of SSP self-assemblies. PMID:27167577

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

    PubMed Central

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-01-01

    Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications. PMID:25756377

  7. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

  8. Structural studies of neuropilin-2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket.

    PubMed

    Tsai, Yi-Chun Isabella; Fotinou, Constantina; Rana, Rohini; Yelland, Tamas; Frankel, Paul; Zachary, Ian; Djordjevic, Snezana

    2016-05-01

    Neuropilin-2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin-2 and its homologous protein neuropilin-1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin-2. The neuropilin-2-specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X-ray crystal structure of the neuropilin-2 b1 domain in a complex with the C-terminal sub-domain of VEGF-A. Zn(2+) binding to neuropilin-2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin-2 ligand binding and signalling activity. PMID:26991001

  9. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  10. A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics

    PubMed Central

    Chaikuad, Apirat; Tacconi, Eliana; Zimmer, Jutta; Liang, Yanke; Gray, Nathanael S.; Tarsounas, Madalena; Knapp, Stefan

    2014-01-01

    Activation of the ERK pathway is a hallmark of cancer and targeting of upstream signalling partners led to the development of approved drugs. Recently SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induced a so far unknown binding pocket that accommodated the piperazine-phenyl-pyrimidine decoration. This novel binding pocket was created by an inactive conformation of the phosphate binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed canonical but two distinct type-I binding modes. Intriguingly, the novel binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity. PMID:25195011

  11. Defining the Plasticity of Transcription Factor Binding Sites by Deconstructing DNA Consensus Sequences: The PhoP-Binding Sites among Gamma/Enterobacteria

    PubMed Central

    Harari, Oscar; Park, Sun-Yang; Huang, Henry; Groisman, Eduardo A.; Zwir, Igor

    2010-01-01

    Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs) using a machine learning method inspired by the “Divide & Conquer” strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target genes and/or the

  12. Binding activities of non-β-glucan glycoclusters to dectin-1 and exploration of their binding site.

    PubMed

    Jiang, Shan; Niu, Shan; Yao, Wang; Li, Zhong-Jun; Li, Qing

    2016-06-24

    Dectin-1, which specifically recognizes β-(1,3)-glucans, plays an important role in innate immune responses. For the first time, in this study we found that a series of non-β-glucan glycoclusters can bind to dectin-1 by means of surface plasmon resonance (SPR) assay. Hexavalent lactoside Ju-6 showed the strongest affinity property (KD=1.6 µM). Interestingly, a continuous binding-dissociation experiment on SPR showed that Ju-6 and Laminarin binding to dectin-1 are independent of each other. Moreover, RT-PCR assay showed that Ju-6 cannot up-regulate cytokine gene expression or inhibit the promoting effect caused by Zymosan (a long-chain β-glucan). These results indicated that there might be a possible new carbohydrate binding site on dectin-1. PMID:27197693

  13. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  14. The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins.

    PubMed

    Jordanova, Rositsa; Radoslavov, Georgi; Fischer, Peter; Torda, Andrew; Lottspeich, Friedrich; Boteva, Raina; Walter, Rolf D; Bankov, Ilia; Liebau, Eva

    2005-12-16

    Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species. PMID:16210327

  15. Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.

    PubMed Central

    Mayo, K H; Ilyina, E; Roongta, V; Dundas, M; Joseph, J; Lai, C K; Maione, T; Daly, T J

    1995-01-01

    Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process. Images Figure 4 PMID:8526843

  16. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

    SciTech Connect

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-08-31

    Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

  17. Computational investigation of stoichiometric effects, binding site heterogeneities, and selectivities of molecularly imprinted polymers.

    PubMed

    Terracina, Jacob J; Bergkvist, Magnus; Sharfstein, Susan T

    2016-06-01

    A series of quantum mechanical (QM) computational optimizations of molecularly imprinted polymer (MIP) systems were used to determine optimal monomer-to-target ratios. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (3-7 molecules) and larger-scale models (15-35 molecules). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to quantify the heterogeneity of these sites. The more fully surrounded sites had greater binding energies. No discretization of binding modes was seen, furthering arguments for continuous affinity distribution models. Molecular mechanical (MM) docking was then used to measure the selectivities of the QM-optimized binding sites. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. Here we present a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Modeling schemes were designed such that no computing clusters or other specialized modeling equipment would be required. Improving the in silico analysis of MIP system properties will ultimately allow for the production of more sensitive and selective polymers. PMID:27207254

  18. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans.

    PubMed

    Weyer, Sven; Pääbo, Svante

    2016-02-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  19. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  20. Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

    PubMed Central

    Wenk, M R; Alt, T; Seelig, A; Seelig, J

    1997-01-01

    The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact

  1. Distribution of cholecystokinin receptor binding sites in the human brain: an autoradiographic study

    SciTech Connect

    Dietl, M.M.; Probst, A.; Palacios, J.M.

    1987-01-01

    Cholecystokinin (CCK) binding sites were localized by in vitro autoradiography in human postmortem brain materials from 12 patients without reported neurological diseases using (125I)Bolton-Hunter CCK octapeptide (BHCCK-8) as a ligand. The pharmacological characteristics of BHCCK-8 binding to mounted tissue sections were comparable to those previously reported in the rat. CCK-8 being the most potent displacer, followed by caerulein, CCK-4, and gastrin I. The distribution of BHCCK-8 binding sites was heterogeneous. These sites were highly concentrated in a limited number of gray matter areas and nuclei. The highest binding densities were seen in the glomerular and external plexiform layers of the olfactory bulb. BHCCK-8 binding sites were also enriched in the neocortex, where they presented a laminar distribution with low levels in lamina I, moderate concentration in laminae II to IV, high density in lamina V, and low levels in lamina VI. A different laminar distribution was seen in the visual cortex, where a low receptor density was observed in lamina IV but higher density in laminae II and VI. In the basal ganglia the nucleus accumbens, caudatus, and the putamen presented moderate to high densities of binding sites, while the globus pallidus lacked sites of BHCCK-8 binding. In the limbic system the only area presenting moderate to high density was the amygdaloid complex, particularly in the granular nucleus, while most of the thalamic nuclei were extremely poor or lacked BHCCK-8 binding. The hippocampal formation showed low (CA1-3) to moderate (subiculum) densities. Midbrain areas generally disclosed very low levels of BHCCK-8 binding sites. The pontine gray and the nucleus reticularis tegmenti pontis showed a relatively high density of CCK-8 receptor specific binding.

  2. A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

    PubMed Central

    Ferrari, Annette; Rijnbrand, R.; Erwin, Alice L.

    2015-01-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux. PMID:25645845

  3. Quantitative autoradiographic distribution of L-(3H)glutamate-binding sites in rat central nervous system

    SciTech Connect

    Greenamyre, J.T.; Young, A.B.; Penney, J.B.

    1984-08-01

    Quantitative autoradiography was used to determine the distribution of L-(3H)glutamate-binding sites in the rat central nervous system. Autoradiography was carried out in the presence of Cl- and Ca2+ ions. Scatchard plots and Hill coefficients of glutamate binding suggested that glutamate was interacting with a single population of sites having a K-D of about 300 nM and a capacity of 14.5 pmol/mg of protein. In displacement studies, ibotenate also appeared to bind to a single class of non-interacting sites with a KI of 28 microM. However, quisqualate displacement of (3H)glutamate binding revealed two well-resolved sites with KIS of 12 nM and 114 microM in striatum. These sites were unevenly distributed, representing different proportions of specific glutamate binding in different brain regions. The distribution of glutamate-binding sites correlated very well with the projection areas of putative glutamatergic pathways. This technique provides an extremely sensitive assay which can be used to gather detailed pharmacological and anatomical information about L-(3H)glutamate binding in the central nervous system.

  4. Sulfhydryl Binding Sites within Bacterial Extracellular Polymeric Substances.

    PubMed

    Yu, Qiang; Fein, Jeremy B

    2016-06-01

    In this study, the concentration of sulfhydryl sites on bacterial biomass samples with and without extracellular polymeric substances (EPS) was measured in order to determine the distribution of sulfhydryl sites on bacteria. Three different approaches were employed for EPS removal from Pseudomonas putida, and the measured sulfhydryl concentrations on bacterial EPS molecules are independent of the EPS removal protocols used. Prior to EPS removal, the measured sulfhydryl sites within P. putida samples was 34.9 ± 9.5 μmol/g, and no sulfhydryl sites were detected after EPS removal, indicating that virtually all of the sulfhydryl sites are located on the EPS molecules produced by P. putida. In contrast, the sulfhydryl sites within the S. oneidensis samples increased from 32.6 ± 3.6 μmol/g to 51.9 ± 7.2 μmol/g after EPS removal, indicating that the EPS produced by S. oneidensis contained fewer sulfhydryl sites than those present on the untreated cells. This study suggests that the sulfhydryl concentrations on EPS molecules may vary significantly from one bacterial species to another, thus it is crucial to quantify the concentration of sulfhydryl sites on EPS molecules of other bacterial species in order to determine the effect of bacterial EPS on metal cycling in the environment. PMID:27177017

  5. The binding sites for cocaine and dopamine in the dopamine transporter overlap

    PubMed Central

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L; Shi, Lei; Gracia, Luis; Raniszewska, Klaudia; Newman, Amy Hauck; Javitch, Jonathan A; Weinstein, Harel; Gether, Ulrik; Loland, Claus J

    2009-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine. PMID:18568020

  6. Binding sites for atrial natriuretic factor (ANF) in brain: alterations in Brattleboro rats

    SciTech Connect

    McCarty, R.; Plunkett, L.M.

    1986-12-01

    Binding sites for atrial natriuretic factor (ANF-28) were analyzed in discrete brain areas of Brattleboro rats with hereditary diabetes insipidus and Long-Evans (LE) controls by quantitative autoradiography. The maximum binding capacity (Bmax) and affinity constant (Ka) for /sup 125/I-ANF-28 were elevated significantly in the subfornical organ of Brattleboro rats compared to matched LE controls. In contrast, values for Bmax and Ka for /sup 125/I-ANF-28 binding in choroid plexus and area postrema were similar for rats of the two strains. These findings are consistent with a selective upregulation of ANF-28 binding sites in the subfornical organ of Brattleboro rats which exhibit a profound disturbance in body fluid homeostasis. These alterations in ANF-28 binding sites in the subfornical organ may represent a compensatory response to the absence of vasopressin in the Brattleboro rat.

  7. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  8. Internal binding sites for MSH: Analyses in wild-type and variant Cloudman melanoma cells

    SciTech Connect

    Orlow, S.J.; Hotchkiss, S.; Pawelek, J.M. )

    1990-01-01

    Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient and centrifuged; fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.

  9. Nanoparticle amplification via photothermal unveiling of cryptic collagen binding sites

    PubMed Central

    Lo, Justin H.; von Maltzahn, Geoffrey; Douglass, Jacqueline; Park, Ji-Ho; Sailor, Michael J.; Ruoslahti, Erkki

    2013-01-01

    The success of nanoparticle-based cancer therapies ultimately depends on their ability to selectively and efficiently accumulate in regions of disease. Outfitting nanoparticles to actively target tumor-specific markers has improved specificity, yet it remains a challenge to amass adequate therapy in a selective manner. To help address this challenge, we have developed a mechanism of nanoparticle amplification based on stigmergic (environment-modifying) signalling, in which a “Signalling” population of gold nanorods induces localized unveiling of cryptic collagen epitopes, which are in turn targeted by “Responding” nanoparticles bearing gelatin-binding fibronectin fragments. We demonstrate that this two-particle system results in significantly increased, selective recruitment of responding particles. Such amplification strategies have the potential to overcome limitations associated with single-particle targeting by leveraging the capacity of nanoparticles to interact with their environment to create abundant new binding motifs. PMID:24177171

  10. Photoaffinity studies of the tubulin-colchicine binding site

    SciTech Connect

    Hahn, K.M.

    1987-01-01

    A variety of colchicine derivatives were synthesized and coupled with 3,3,3-trifluoro-2-diazapropionyl chloride (TFDP-Cl) to produce colchicine photoaffinity analogs for use in tubulin labelling studies. Photoaffinity analogs of allocolchicine and podophylotoxin were also made using the same photoreactive moiety. Several labels were found to be effective inhibitors of tubulin polymerization. The approximate tubulin binding constants of the labels, calculated from polymerization inhibition data, varied between 2.2 x 10/sup 5/ to 2.5 x 10/sup 3/ M/sup -1/. The labels chosen for use in tubulin labelling experiments were (N-TFDP) deacetyl-thiocolchicine 1, (O-TFDP)thiocolchifoline 2, and (O-TFDP)-2-demethylthiocolchicine 3. Compound 1 was found to bind tubulin reversibly and to competitively inhibit colchicine binding. Methods for the incorporation of tritium and /sup 14/C in these labels were developed. Conditions were found which caused labels to insert into solvent without photorearrangement of the colchicine skeleton. Catalytic base caused the ..cap alpha..-diazo amide of 1 to rearrange to a triazole.

  11. Direct binding of triglyceride to fat storage-inducing transmembrane proteins 1 and 2 is important for lipid droplet formation.

    PubMed

    Gross, David A; Zhan, Chenyang; Silver, David L

    2011-12-01

    The process of lipid droplet (LD) formation is an evolutionarily conserved process among all eukaryotes and plays an important role in both cellular physiology and disease. Recently, fat storage-inducing transmembrane proteins 1 and 2 (FIT1/FITM1 and FIT2/FITM2) were discovered as an evolutionarily conserved family of proteins involved in fat storage. In mammals, FIT1 is expressed primarily in skeletal muscle and FIT2 is expressed primarily in adipose, raising the possibility that FIT1 and FIT2 have unique functions. These proteins are exclusively localized to the endoplasmic reticulum (ER) and mediate triglyceride-rich LD accumulation when overexpressed in cells, mouse liver, or muscle. Unlike the ER-resident diacylglycerol O-acyltransferase family of triglyceride-synthesizing enzymes, FITs do not synthesize triglyceride, but rather partition triglyceride into LDs. The mechanism by which FIT proteins mediate this process has not been determined. A simple hypothesis was tested that FIT proteins bind to triglyceride to mediate LD formation. Here, it is shown that FIT proteins purified in detergent micelles directly bind triolein with specificity and saturation-binding kinetics. A FIT2 gain-of-function mutant that formed larger LDs, FLL(157-9)AAA, showed increased binding to triolein relative to wild-type FIT2, whereas FIT1 and a FIT2 partial loss-of-function mutant, N80A, had significantly lower triolein binding and produced smaller LDs. In summary, FIT proteins are transmembrane domain-containing proteins shown to bind triglyceride. These findings indicate that FITs have a unique biochemical mechanism in mediating LD formation and implicates triglyceride binding as important for FIT-mediated LD formation. PMID:22106267

  12. Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome

    PubMed Central

    Dresch, Jacqueline M.; Zellers, Rowan G.; Bork, Daniel K.; Drewell, Robert A.

    2016-01-01

    A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development. PMID:27330274

  13. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-08-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7659518

  14. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-09-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7567442

  15. Identification of binding sites for the group A streptococcal global regulator CovR.

    PubMed

    Federle, Michael J; Scott, June R

    2002-03-01

    The CovRS two-component system (also called CsrRS) of the group A streptococcus (GAS) acts as a global regulator, influencing the transcription of at least six virulence factors. The synthesis of the hyaluronic acid capsule, a virulence factor encoded by the hasABC operon, is negatively regulated by CovRS. We confirmed that phosphorylation of CovR increases its binding to a DNA fragment containing the hasA promoter. Using DNase I footprinting, we identified five binding sites surrounding the hasA promoter from bases -79 to +73 (where +1 is the start of transcription). One pair of thymines within each binding site appears to be necessary for CovR binding in vitro, as shown by uracil interference analysis. When each of these thymine pairs was altered by site-directed mutagenesis, CovR binding was reduced in vitro, confirming the role of each thymine pair in binding. Using a transcriptional reporter system with a single chromosomal copy of PhasA-gusA, we demonstrated the importance of each of four of these binding sites for CovR repression of the hasA promoter. Based on this information, we propose a consensus sequence for CovR binding to DNA. PMID:11918804

  16. Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites

    PubMed Central

    Smith, Amy Rhoden; Iverson, Brent L.

    2013-01-01

    The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-visible spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators. PMID:23919778

  17. Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites.

    PubMed

    Rhoden Smith, Amy; Iverson, Brent L

    2013-08-28

    The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-vis spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid-based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators. PMID:23919778

  18. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

  19. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

    PubMed

    Goto, Asako; Charman, Mark; Ridgway, Neale D

    2016-01-15

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

  20. DNA binding site characterization by means of Rényi entropy measures on nucleotide transitions.

    PubMed

    Perera, A; Vallverdu, M; Claria, F; Soria, J M; Caminal, P

    2008-06-01

    In this work, parametric information-theory measures for the characterization of binding sites in DNA are extended with the use of transitional probabilities on the sequence. We propose the use of parametric uncertainty measures such as Rényi entropies obtained from the transition probabilities for the study of the binding sites, in addition to nucleotide frequency-based Rényi measures. Results are reported in this work comparing transition frequencies (i.e., dinucleotides) and base frequencies for Shannon and parametric Rényi entropies for a number of binding sites found in E. Coli, lambda and T7 organisms. We observe that the information provided by both approaches is not redundant. Furthermore, under the presence of noise in the binding site matrix we observe overall improved robustness of nucleotide transition-based algorithms when compared with nucleotide frequency-based method. PMID:18556261

  1. An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site

    PubMed Central

    Lu, Yan; Chen, Jianjun; Xiao, Min; Li, Wei

    2013-01-01

    Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs. PMID:22814904

  2. Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites.

    PubMed

    Chaloupkova, Radka; Prudnikova, Tatyana; Rezacova, Pavlina; Prokop, Zbynek; Koudelakova, Tana; Daniel, Lukas; Brezovsky, Jan; Ikeda-Ohtsubo, Wakako; Sato, Yukari; Kuty, Michal; Nagata, Yuji; Kuta Smatanova, Ivana; Damborsky, Jiri

    2014-07-01

    The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine. PMID:25004965

  3. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

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

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  4. Brain natriuretic peptide binding sites in rats: In vitro autoradiographic study

    SciTech Connect

    Konrad, E.M.; Thibault, G.; Pelletier, S.; Genest, J.; Cantin, M. )

    1990-08-01

    Brain natriuretic peptide (BNP) is a recently discovered family of natriuretic peptides highly homologous to atrial natriuretic factor (ANF). Quantitative in vitro autoradiography with a computerized microdensitometer demonstrated that the distribution of BNP binding sites is similar to the known distribution pattern of ANF binding sites in rat tissues. Analysis of saturation and competition curves disclosed that the maximal binding capacity for BNP-(Asp-81--Tyr-106) and ANF-(Ser-99--Tyr-126) is similar within the plexiform layer of the olfactory bulb, the choroid plexus, and the adrenal zona glomerulosa. Examination of the competition curves of BNP-(Asp-81--Tyr-106), ANF-(Ser-99--Tyr-126), and des-(Gln-116--Gly-120)ANF-(Asp-102--Cys-121)NH2 (C-ANF, a ligand highly specific for ANF-R2 receptors) for {sup 125}I-labeled BNP-(Asp-81--Tyr-106) and {sup 125}I-labeled ANF-(Ser-99--Tyr-126) binding revealed that ANF fully displaced {sup 125}I-BNP binding and, conversely, BNP completely displaced {sup 125}I-ANF binding in these tissues, whereas C-ANF partially displaced 125-BNP and 125-ANF binding. Angiotensin II, insulin, glucagon, and substance P had no influence on {sup 125}I-BNP binding in the above tissues. These results support the view that BNP and ANF share the same binding sites in rats.

  5. Dual Effects of Adp and Adenylylimidodiphosphate on Cftr Channel Kinetics Show Binding to Two Different Nucleotide Binding Sites

    PubMed Central

    Weinreich, Frank; Riordan, John R.; Nagel, Georg

    1999-01-01

    The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of pa