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Showing papers on "Cooperative binding published in 2001"


Journal ArticleDOI
TL;DR: It is proposed that sequential recognition of high‐ and low‐affinity sites, and binding to single‐stranded origin DNA may be general properties of initiator proteins in initiation complexes.
Abstract: The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5'-TT(A/T)TNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP-DnaA box (5'-AGatct). Using DNase footprinting and surface plasmon resonance we show that binding to ATP-DnaA boxes in the AT-rich region of oriC of E.coli requires binding to the 9mer DnaA box R1. Cooperative binding of ATP-DnaA to the AT-rich region results in its unwinding. ATP-DnaA subsequently binds to the single-stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single-stranded ATP-DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were approximately 1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA boxes, respectively. We propose that sequential recognition of high- and low-affinity sites, and binding to single-stranded origin DNA may be general properties of initiator proteins in initiation complexes.

204 citations


Journal ArticleDOI
TL;DR: The results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells and support the effect of NaCl concentration on the mode of binding of poly-l-lysine to DNA.

159 citations


Journal ArticleDOI
TL;DR: In protein NMR relaxation data, changes in flexibility that occur upon ligand binding, mutation, or changes in sample conditions can be interpreted in terms of contributions to conformational entropy as mentioned in this paper.
Abstract: Recent advances in the measurement and analysis of protein NMR relaxation data have made it possible to characterize the dynamical properties of many backbone and side chain groups. With certain caveats, changes in flexibility that occur upon ligand binding, mutation, or changes in sample conditions can be interpreted in terms of contributions to conformational entropy. Backbone and side chain flexibility can either decrease or increase upon ligand binding. Decreases are often associated with “enthalpy−entropy compensation” and “induced fit” binding, whereas increases in conformational entropy can contribute to stabilization of complexes. In certain cases, conformational entropy appears to play a role in cooperative binding and enzyme catalysis. In addition, variations in conformational entropy and heat capacity may both be important in stabilizing the folded structures of proteins.

152 citations


Journal ArticleDOI
TL;DR: A novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated is presented, which takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations.
Abstract: Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from a ∼100-fold improvement (7.4 nM) to a ∼two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).

147 citations


Journal ArticleDOI
TL;DR: Unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.
Abstract: The structure of beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit. The comparison of the E. coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein. This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer. FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.

139 citations


Journal ArticleDOI
TL;DR: The substituted cysteine accessibility method was used to identify binding pocket residues but also to elicit information about binding site dynamics and structure, suggesting that the binding site may constrict during gating.
Abstract: Photo-affinity labeling and mutagenesis studies have identified several amino acids that may contribute to the ligand binding domains of ligand-gated ion channels. These types of studies, however, only generate a one-dimensional, static description of binding site structure. In this study, we used the substituted cysteine accessibility method not only to identify binding pocket residues but also to elicit information about binding site dynamics and structure. Residues surrounding the putative loop C ligand binding domain of the GABAA receptor (β2V199 to β2S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type α1subunits in Xenopus oocytes. N -biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an α-helix or β-strand. Instead, G203–S209 seems to form a water-accessible extended coil, whereas V199–T202 appears to buried in the protein or membrane. Coapplication of either GABA or the competitive antagonist SR-95531 significantly slows MTSEA-biotin modification of cysteines introduced at positions S204, Y205, R207, and S209, demonstrating that these residues line and face into the GABA binding pocket. MTSEA-biotin reaction rates reveal a steep accessibility gradient from G203–S209 and suggests that the binding pocket is a deep narrowing cleft. Pentobarbital activation of the receptor significantly slows MTSEA-biotin modification of cysteines at S204, R207, and S209, suggesting that the binding site may constrict during gating.

138 citations


Journal ArticleDOI
25 Oct 2001-Neuron
TL;DR: The superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

126 citations


Journal ArticleDOI
TL;DR: Results suggested that the binding site(s) for the CBD is(are) present in the collagenous region and specifically recognizes the triple-helical conformation made by three polypeptide chains in thecollagenous region.

120 citations


Journal ArticleDOI
TL;DR: ExAFS spectroscopy of the mono- and di-Zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data and shows negative cooperativity.

118 citations


Journal ArticleDOI
TL;DR: Although similar regions of X4 and R5 HIV-1 gp120s appear to be involved in binding CXCR4 and CCR5, respectively, differences exist in nonspecific binding to cell surfaces, affinity for the chemokine receptor, and CD4 dependence at low temperature.

106 citations


Journal ArticleDOI
TL;DR: The results suggest that alteration of the affinity of the vinblastine binding site involves only one nucleotide binding domain per transport cycle.
Abstract: Conceptually one may envisage that substrate binding sites on the ABC transporter P-gp cycle between high- and low-affinity conformations in response to signals arising from nucleotide hydrolysis to effect active transport. A radioligand binding assay was used to characterize the interaction of [3H]vinblastine with P-gp and determine how drug binding site parameters are altered during a catalytic cycle of P-gp. In the absence of nucleotide, we show that [3H]vinblastine interacts with a single class of binding site with high affinity (K(d) = 80 +/- 18 nM). In the presence of the nonhydrolyzable ATP analogue AMP-PNP, the drug binding site was in a low-affinity conformation, manifest by a 9-fold increase in K(d) (K(d) = 731 +/- 20 nM). There was no alteration in the binding capacity, reflecting a complete shift in the high-affinity site to a low-affinity form. The posthydrolytic (Mg-ADP-V(i) bound) form of P-gp also exhibited low-affinity substrate binding (K(d) = 446 +/- 57 nM). Restoration of the high-affinity drug binding site conformation (K(d) = 131 +/- 32 nM) did not occur until release of phosphate from the posthydrolysis P-gp-Mg-ADP-P(i). complex. Our results suggest that alteration of the affinity of the vinblastine binding site involves only one nucleotide binding domain per transport cycle. The binding of ATP provides the signal to instigate this change, while release of phosphate post-ATP hydrolysis returns the transporter to its original state to complete the cycle.

Journal ArticleDOI
TL;DR: Using NMR to study the allosteric mechanism of this enzyme, widespread chemical shift changes for the individual CTP binding steps are observed, suggesting that a fraction of the free energy of negative cooperativity is entropic in origin.
Abstract: The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.

Journal ArticleDOI
TL;DR: Using synthetic peptides, it is shown that the γ subunit binds to two distinct sites on the catalytic αβ dimer when the regulatory GAF domains of bovine rod PDE6 are occupied by cGMP.

Journal ArticleDOI
TL;DR: Binding of eIF4A to an immobilized fragment of eif4G containing the COOH-terminal site was competed by a soluble eIF2G fragment containing the central site, indicating that a single eIF 4A molecule cannot bind simultaneously to both sites.

Journal ArticleDOI
TL;DR: It is demonstrated that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p‐Gal80p interaction and also, indirectly, the Gal 4p‐DNA interaction in a (Gal4p)2(Gal80 p)2DNA complex.
Abstract: Regulation of the GAL genes of Saccharomyces cerevisiae is determined by the interplay of the transcriptional activator Gal4p and the repressor Gal80p, which binds and masks the activation domain of Gal4p under non-inducing conditions. Here we demonstrate that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p-Gal80p interaction and also, indirectly, the Gal4p-DNA interaction in a (Gal4p)2(Gal80p)2DNA complex. In addition, Gal80 dimers transiently interact with each other to form higher order multimers. We provide evidence that adjacent Gal4p binding sites, when correctly spaced, greatly stabilize Gal80p dimer-dimer interactions and that this stabilization results in the complete repression of GAL genes with multiple Gal4p binding sites. In contrast, GAL genes under the control of a single Gal4p binding site do not stabilize Gal80p multimers, resulting in significant and biologically important transcriptional leakage. Cooperative binding experiments indicate that Gal80p dimer-dimer interaction probably does not lead to a stronger Gal4p-Gal80p interaction, but most likely to a more complete shielding of the Gal4p activation domain.

Journal ArticleDOI
TL;DR: This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.
Abstract: The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.

Journal ArticleDOI
TL;DR: A novel analytic isotherm is derived that develops an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecial binding sites.

Journal ArticleDOI
20 Dec 2001-Neuron
TL;DR: Three-dimensional modeling indicates that localized regions in the S1 binding domain of both subunits required for the transmission of allosteric signals from the glutamate binding NR2A subunit to the glycine binding NR1 subunit mediate negative allosterics coupling between the two subunit types that form the NMDA receptor.

Journal ArticleDOI
TL;DR: The mechanism of the cooperative binding of S6 and S18 to the S15-rRNA complex is studied by isothermal titration calorimetry and gel mobility shift assays with rRNA and proteins from the hyperthermophilic bacterium Aquifex aeolicus to provide a mechanistic framework to describe the previously observed S15.

Journal ArticleDOI
TL;DR: NMR and emission titration studies with receptors 1-4 and KCl/KOAc show that cobound potassium enhances anion binding strength by electrostatic and conformational effects.
Abstract: The new rhenium(I) bipyridine crown ether receptors 1-4 have been prepared and their ion pair recognition properties examined. The crystal structure of [1.KCl](2).2H(2)O demonstrates that potassium is coordinated by benzo-18-crown-6 and chloride is hydrogen bonded to the amide groups. Receptor 3 extracts solid KCl and KOAc into chloroform via ion pair complexation. NMR and emission titration studies with receptors 1-4 and KCl/KOAc show that cobound potassium enhances anion binding strength by electrostatic and conformational effects. Significant cooperative interactions are observed between the anion and cation sites for host 4 in CH(3)CN. This molecule coordinates potassium to form a 1:1 intramolecular sandwich complex, which preorganizes the host for acetate binding.

Journal ArticleDOI
TL;DR: The synthesis and fluorescent properties of a second generation cooperative chemical sensor has two interacting binding pockets for cooperative recognition of two analytes and was characterized by NMR, absorption, and fluorescence spectroscopy.
Abstract: The synthesis and fluorescent properties of a second generation cooperative chemical sensor are described. The sensor has two interacting binding pockets for cooperative recognition of two analytes. Cooperative binding activates a ratiometric fluorescent response via formation of an excimer. Binding was characterized by NMR, absorption, and fluorescence spectroscopy. The advantages of separating the recognition elements from the fluorescent response elements are discussed.

Journal ArticleDOI
TL;DR: Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL 4-dd on GAL 11P interaction.
Abstract: The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P.GAL11P (like GAL11) is a component of the RNA–polymerase II holoenzyme.Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C2 symmetry containing three helices that mediate dimerization via coiled-coil contacts.The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs.Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix 3 and the loop between 1 and 2.One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction.The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.

Journal ArticleDOI
TL;DR: Using gel mobility shift and fluorescence anisotropy assays, it is determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the HSp70·RNA complex.

Journal ArticleDOI
TL;DR: The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding.

Journal ArticleDOI
TL;DR: A rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations, and alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected.
Abstract: A variety of theoretical methods including classical molecular interaction potentials, classical molecular dynamics, and activated molecular dynamics have been used to analyze the substrate recognition mechanisms of peroxisomal catalase from Saccharomyces cerevisiae. Special attention is paid to the existence of channels connecting the heme group with the exterior of the protein. On the basis of these calculations a rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations. According to our calculations the water is expected to be a competitive inhibitor of the enzyme, blocking the access of hydrogen peroxide to the active site. The main channel is the preferred route for substrate access to the enzyme and shows a cooperative binding to hydrogen peroxide. However, the overall affinity of the main channel for H(2)O(2) is only slightly larger than that for H(2)O. Alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected. These secondary channels might be important for product release.

Journal ArticleDOI
TL;DR: The binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL and the fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other.

Journal ArticleDOI
TL;DR: X-ray crystallography and small angle X-ray scattering in solution is used to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.
Abstract: Regulation of protein function, often achieved by allosteric mechanisms, is central to normal physiology and cellular processes. Although numerous models have been proposed to account for the cooperative binding of ligands to allosteric proteins and enzymes, direct structural support has been lacking. Here, we used a combination of X-ray crystallography and small angle X-ray scattering in solution to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.

Journal Article
TL;DR: A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site, leading to a model of the changes in the binding sites caused by ligand binding.
Abstract: A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.

Journal Article
TL;DR: Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors, and reveal independence between mutation-induced changes in the affinity for a modulator and inThe magnitude and direction of the allosteric effect of the modulator.
Abstract: To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.

Journal ArticleDOI
TL;DR: A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs.
Abstract: A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.