Showing papers on "Cooperative binding published in 1992"

Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding.

Abstract: The CACGTG G-box motif is a highly conserved DNA sequence that has been identified in the 5' upstream region of plant genes exhibiting regulation by a variety of environmental signals and physiological cues. Gel mobility shift assays using a panel of G-box oligonucleotides differing in their flanking sequences identified two types of binding activity (A and B) in a cauliflower nuclear extract. Competition gel retardation assays demonstrated that the two types of binding activity were distinct. Type A binding activity interacted with oligonucleotides designated as class I elements, whereas type B binding activity interacted strongly with class II elements and weakly with class I elements. A third class of elements, null elements, did not exhibit any detectable binding under our assay conditions. Gel retardation analysis of nonpalindromic hybrid G-box oligonucleotides indicated that hybrid elements of the same class exhibited binding affinity commensurate with the affinity of the weaker element, hybrid class I/II elements exhibited only type B binding, and hybrid class I/null and class II/null elements did not show any detectable binding activity. These binding activities can be explained by the affinity of bZip G-box binding homo- or heterodimer subunits for G-box half sites. These experiments led to a set of classification rules that can predict the binding activity of all reported plant G-box motifs containing the consensus hexameric core. Tissue- and/or development-specific expression of genes containing G-box motifs may be regulated by the affinity of G-box proteins for the different classes of G-box elements.

Protein-induced bending and DNA cyclization

Abstract: We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.

Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription

Abstract: We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Both phosphorylated and unphosphorylated NRI bind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NRI is severely affected by phosphorylation: half-maximal binding of NRI-phosphate is at 20-fold lower concentrations than that of unphosphorylated NRI. This is more due to a huge increase in the cooperativity constant--which is the strength of interaction between two NRI dimers--than to an increase in the microscopic binding constant which is the binding affinity to a single binding site. In vitro transcription and DNA footprinting experiments showed that occupation of a single binding site by NRI is not enough for efficient activation and that activation only occurs at a higher NRI concentration. We propose an activation mechanism for NRI in which the phosphorylation of NRI induces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NRI upon binding to two adjacent binding sites. We propose that not the phosphorylation of NRI itself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

Abstract: The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.

Cooperative binding of the red clover necrotic mosaic virus movement protein to single-stranded nucleic acids.

Abstract: The movement protein of red clover necrotic mosaic dianthovirus was produced in Escherichia coli using an expression vector. Gel retardation analysis and u.v. cross-linking studies showed that the movement protein bound cooperatively to ssRNA and ssDNA, but not to dsDNA. Binding competition experiments established that the movement protein bound to ssRNA and ssDNA with similar affinities and that the binding was not sequence-specific in the experimental conditions employed. A truncated movement protein lacking the C-terminal 88 amino acids was also shown to bind to ssRNA.

Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor.

Abstract: Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.

Cooperative binding of the globular domains of histones H1 and H5 to DNA

Abstract: In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.

Capacity for Cooperative Binding of Thyroid Hormone (T3) Receptor Dimers Defines Wild Type T3 Response Elements

Abstract: Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Triple-helix formation and cooperative binding by oligodeoxynucleotides with a 3'-3' internucleotide junction.

Abstract: Triple-helix formation by oligodeoxynucleotides in a sequence-specific manner is limited to polypurine tracts of duplex DNA. To increase the number of biologically relevant targets for triple-helix formation, we have utilized oligodeoxynucleotides containing a 3'-3' internucleotide junction to allow for binding to opposite strands of duplex DNA. Molecular modeling was used to aid in the design of the xylose dinucleoside linker 1 that is rigid and minimizes the number of conformers to minimize the entropy of binding. Thermal denaturation studies show that a 3'-3'-linked oligodeoxynucleotide, bearing nine nucleotides on each side of the linker, has a higher Tm (47.6 degrees C) than that of a 21-mer binding to a single polypurine tract (45.3 degrees C). Binding domain minimization studies and sequence-specific alkylation of a target duplex demonstrate a high degree of cooperativity between the two triple-helix binding domains, thus allowing for an increase in the number of biologically relevant targets for triple-helix formation.

Evidence that drug binding to non-adrenergic [3H]-idazoxan binding sites (I-receptors) occurs to interacting or interconvertible affinity forms of the receptor.

Abstract: Characterization of [3H]idazoxan binding to guinea pig kidney membranes showed that approximately 90% bound to nonadrenergic I-receptors and approximately 10% to alpha 2-adrenoceptors. I-Receptors could be studied separately by including 3 microM rauwolscine to the assay. During these conditions 22 different compounds out of 29 (including imidazoline and guanidinium compounds) generated biphasic or shallow competion curves (Hill coefficients down to 0.57), for which computer modelling suggested that drugs bound to two sites with different affinities. However, the proportion of sites varied considerably depending on which drug was used as competitor; the variation being from approximately 50/50% to approximately 8/92% and analysis of variance clearly indicated that the variation of proportions of sites could be attributed to an effect induced by the drugs which indicated that the sites were dynamically formed or modulated by the presence of the drug. A few drugs (guanabenz, (-)-medetomidine, phentolamine, clemastin, prazosin and idazoxan itself) yielded steep uniphasic curves (Hill coefficients near unity) which were resolved only into one site fits. One drug (detomidine) yielded supersteep competition curves (Hill coefficient 1.29), the data being reminiscent of positive cooperativity. In another set of experiments attempts were made to block one of the affinity forms of the I-receptor with histamine, a compound which had grossly different affinities for the two I-receptor sites, and competition curves were then obtained using other drugs which also seemed to distinguish between the two sites. Computer analysis of experimentally obtained as well as simulate,d computer generated, competition curves clearly showed that the kidney I-receptors did not exist in two independent non-interacting forms. Instead the data suggested that the I-receptor was composed of interacting or interconverting receptor entities with different affinities for drugs. Monovalent cations such as Cs+ or NH4+ were found to be powerful inhibitors of [3H]idazoxan binding to the kidney I-receptors, the ions decreasing both the apparent Bmax and affinity of the ligand for the receptor. The cations also decreased the affinities of UK-14,304 for both the high and low affinity forms of the I-receptor, but the apparent proportions of sites were not at all affected by the ions. Moreover, ligand binding to kidney I-receptor were not at all affected by non-hydrolyzable guanine nucleotides. It is suggested that the binding data reflects functional properties of the I-receptor protein.

The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor.

Abstract: The mechanism of binding of murine IFN-gamma to its receptor has not been determined. We have studied this mechanism by examining the binding of overlapping synthetic peptides of IFN-gamma to cloned soluble murine IFN-gamma R. IFN-gamma (1-39) and IFN-gamma (95-133) were able to compete with [125I]IFN-gamma for binding to cloned soluble receptor. Peptides corresponding to the inner region of IFN-gamma--IFN-gamma (36-60), IFN-gamma (54-91), and IFN-gamma (78-107)--showed a markedly reduced ability to compete with [125I]IFN-gamma for receptor binding relative to the N-terminal and C-terminal peptides. In direct binding studies, the binding of [125I]-IFN-gamma (1-39) to soluble receptor could only be competed by IFN-gamma (1-39) and IFN-gamma and not by any of the other peptides including IFN-gamma (95-133). This suggests that the N- and C-termini of IFN-gamma bind to different regions of the receptor. These data in conjunction with previous structure/function studies and x-ray crystallographic data have allowed us to formulate a "velcro-key" model of IFN-gamma binding to receptor that involves both the N- and C-terminal domains. The N-terminus binds in the classical "lock-and-key" manner characterized by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct from the N-terminus likely through the polycationic region, which is conserved across species barriers. Binding of this type would exhibit high affinity and low specificity similar to a piece of velcro. This interaction becomes specific when the C-terminus is in the context of the whole IFN-gamma molecule and may act to increase the affinity of receptor binding and/or facilitate signal transduction.

Cooperative Binding of Adenine via Complementary Hydrogen Bonding to an Imide Functionalized Monolayer at the Air-Water Interface

Abstract: Double-chain derivatives of orotic acid (1) and hydroorotic acid (2) formed stable monolayers at the air-water interface. FT-IR and ESCA spectroscopies revealed specific cooperative binding of adenine from the aqueous subphase to monolayer 1 in preference to thymine. Monolayer 2 did not bind these nucleic acid bases.

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Abstract: Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.

Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.

Abstract: A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms. The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.

Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.

Abstract: Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

Abstract: A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.