Showing papers on "Cooperative binding published in 2003"
TL;DR: The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.
118 citations
TL;DR: To systematically define the molecular determinants of FKBP12.6 binding, a series of internal and NH2- and COOH-terminal deletion mutants of RyR2 were constructed and the effect of these deletions on GST-FKBP 12.6binding was examined.
105 citations
TL;DR: It is shown that in the binding of biotin to streptavidin, the streptavin structure becomes better packed, and it is concluded that this contraction of the strePTavidin structure promotes biotin binding.
94 citations
TL;DR: Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.
90 citations
TL;DR: In this paper, the authors show that removing the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap but not to proximal ones.
84 citations
TL;DR: Methods of inhibitor cross-competition kinetics and competition ligand binding were utilized to address whether non-transition state small molecule inhibitors, which also display non-competitive inhibition of γ-secretase, inhibit the enzyme by binding to the catalytic site as well.
78 citations
TL;DR: An investigation into the thermodynamic origin of cooperativity in ion-pairing host-guest binding in water is described, revealing that entropy is the largest contributing factor.
Abstract: The association of synthetic receptors to target guests often proceeds through the cooperative action of multiple binding forces. An investigation into the thermodynamic origin of cooperativity in ion-pairing host−guest binding in water is described. The binding affinities of 1,2,3,4-butanetetracarboxylate, tricarballate, glutarate, and acetate to a C3v symmetric metallo-host (1) are characterized in terms of the binding constants (Ka) and the thermodynamic parameters ΔG°, ΔH°, and ΔS°, as determined by isothermal titration calorimetry (ITC). These values are used to determine the individual contributions of the binding interaction to the overall binding. Several ways to view the combination of the individual binding events that make up the whole are analyzed, all of which lead to the conclusion of negative cooperativity. Combined, the data were used to evaluate the thermodynamic origin of negative cooperativity for this series of guests, revealing that entropy is the largest contributing factor. An inter...
76 citations
TL;DR: Competitive binding experiments were performed using Y1-BS1 adrenal cells to provide information about the interaction of HDL apolipoproteins with scavenger receptor class B, type I (SR-BI), consistent with the existence of more than one type of Apolipoprotein binding site on SR-BI.
73 citations
TL;DR: In this paper, a homology model of the ligand binding domain of the α7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure.
71 citations
TL;DR: A novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists is demonstrated: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG.
Abstract: Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF(1)) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk(-) cells expressing the cloned human CRF(1) receptor. R was detected with the antagonist (125)I-astressin with 30 microM guanosine 5'-O-(3-thiotriphosphate present, and RG detected using (125)I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited (125)I-astressin binding (22-32% maximal inhibition). NBI 35965 accelerated (125)I-astressin dissociation and only partially increased the IC(50) value of unlabeled sauvagine, CRF, and urocortin for displacing (125)I-astressin binding (by 4.0-7.1-fold). Reciprocal effects at the R state were demonstrated using [(3)H]NBI 35965: agonist peptides only partially inhibited binding (by 13-40%) and accelerated [(3)H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited (125)I-sauvagine binding at low radioligand concentrations (79-94 pM). NBI 35965 did not completely inhibit (125)I-sauvagine binding at high radioligand concentrations (82 +/- 1%, 1.3-2.1 nM) and slowed dissociation of (125)I-sauvagine and (125)I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds.
67 citations
TL;DR: The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.
TL;DR: The solution structure of apo-S100A11 has been determined by NMR spectroscopy to uncover the details of its calcium-induced structural change and results in a "masked" binding surface that prevents annexin I binding but is uncovered upon calcium binding.
Journal Article•
TL;DR: The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state, suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions.
Abstract: A homology model of the ligand binding domain of the a7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure. This structure is refined in a 10 ns molecular dynamics simulation. The modeled structure proves fairly resilient, with no significant changes at the secondary or tertiary structural levels. The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state. Candidate motions that take place involve not only the side chains of residues lining the binding sites, but also the subunit positions that determine the overall shape of the receptor. In particular, two nonadjacent subunits move outward, whereas their partners counterclockwise to them move inward, leading to a marginally wider interface between themselves and an overall asymmetric structure. This in turn affects the binding sites, producing two that are more open and characterized by distinct side-chain conformations of W54 and L118, although motions of the side chains of all residues in every binding site still contribute to a reduction in binding site size, especially the outward motion of W148, which hinders acetylcholine binding. The Cys loop at the membrane interface also displays some flexibility. Although the short simulation timescale is unlikely to sample adequately all the conformational states, the pattern of observed motions suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions. Furthermore, the shape of the asymmetry with binding sites of differing affinity for acetylcholine, characteristic of other nicotinic receptors, may be a natural property of the relaxed, activatable state of a7.
TL;DR: It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosterics site modulates receptor-receptor interactions such that homotropic positive cooperateativity within a dimer or higher oligomer is generated.
Abstract: An optimized assay for the binding of [3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the allosteric site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated.
TL;DR: The thermodynamics of formation of a novel divalent metal ion-DNA complex known as M-DNA have been investigated using an ethidium bromide (EB) fluorescence assay, and with isothermal titration calorimetry to show an entropy driven process.
TL;DR: It was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues, which indicates that the Site2 binding interface of the hGH–hGHR ternary complex displays both structural and energetic malleability.
Abstract: Receptor signaling in the growth hormone (GH)–growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single- and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by ≥10 A, this indicates that the Site2 binding interface of the hGH–hGHR ternary complex displays both structural and energetic malleability.
TL;DR: It is concluded that biochemical cooperativity can be mediated through changes in vibrational activity, irrespective of the presence or absence of concomitant structural change.
Abstract: Oligomerization and ligand binding are thermodynamically cooperative processes in many biochemical systems, and the mechanisms giving rise to cooperative behavior are generally attributed to changes in structure. In glycopeptide antibiotics, however, these cooperative processes are not accompanied by significant structural changes. To investigate the mechanism by which cooperativity arises in these compounds, fully solvated molecular dynamics simulations and quasiharmonic normal-mode analysis were performed on chloroeremomycin, vancomycin, and dechlorovancomycin. Configurational entropies were derived from the vibrational modes recovered from ligand-free and ligand-bound forms of the monomeric and dimeric species. Results indicate that both ligand binding and dimerization incur an entropic cost as vibrational activity in the central core of the antibiotic is shifted to higher frequencies with lower amplitudes. Nevertheless, ligand binding and dimerization are cooperative because the entropic cost of both processes occurring together is less than the cost of these processes occurring separately. These reductions in configurational entropy are more than sufficient in magnitude to account for the experimentally observed cooperativity between dimerization and ligand binding. We conclude that biochemical cooperativity can be mediated through changes in vibrational activity, irrespective of the presence or absence of concomitant structural change. This may represent a general mechanism of allostery underlying cooperative phenomena in diverse macromolecular systems.
TL;DR: Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding.
TL;DR: Structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
TL;DR: Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, this work has identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system.
TL;DR: The relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.
TL;DR: A complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate, is described.
TL;DR: The results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosterics behavior arises from coupled tertiary conformational changes.
Abstract: The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 → Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 → Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.
TL;DR: Using tethering, surface plasmon resonance (SPR), and X-ray crystallography, it is discovered that the IL-2 adaptive region contains at least two cooperative binding sites where thebinding of a first ligand to one site promotes or antagonizes the binding of a second ligands to the second site.
Abstract: The cytokine hormone interleukin-2 (IL-2) contains a highly adaptive region that binds small, druglike molecules The binding properties of this adaptive region have been explored using a "tethering" method that relies on the formation of a disulfide bond between the protein and small-molecule ligands Using tethering, surface plasmon resonance (SPR), and X-ray crystallography, we have discovered that the IL-2 adaptive region contains at least two cooperative binding sites where the binding of a first ligand to one site promotes or antagonizes the binding of a second ligand to the second site Cooperative energies of interaction of -2 kcal/mol are observed The observation that the adaptive region contains two adjacent sites may lead to the development of tight-binding antagonists of a protein-protein interaction Cooperative ligand binding in the adaptive region of IL-2 underscores the importance of protein dynamics in molecular recognition The tethering approach provides a novel and general strategy for discovering such cooperative binding interactions in specific, flexible regions of protein structure
TL;DR: The authors' data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3) and the relevance of such interactions during prion pathogenesis remains to be established.
TL;DR: The energetics of calcium binding and the strong relationship between calcium binding cooperativity and conformational change are discussed and "grafting" is introduced as a novel approach to obtain the site-specific metal binding properties of calmodulin.
Abstract: Calmodulin (CaM) is an EF-hand Ca(II)-binding protein involved in the regulation of many important biological processes. To date, there is a wealth of information available concerning studies to obtain site-specific calcium binding affinities of CaM, and further to estimate the cooperativity of calcium binding using mutational studies, peptide models, and proteolytic fragmentation. In this paper, we will discuss the energetics of calcium binding and the strong relationship between calcium binding cooperativity and conformational change. We then explain the difficulty of studying key determinants of calcium binding affinity of CaM due to the large change of calcium binding affinity upon mutation. Subsequently, we will introduce “grafting” as a novel approach to obtain the site-specific metal binding properties of calmodulin.
TL;DR: To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor, glutamate amino acid residues at three different positions in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis.
TL;DR: There is a decrease in responsiveness to exogenous 1,25-dihydroxyvitamin D(3) as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1, 25-(OH)(2)D(3), a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.
Abstract: To study the physiological relevance of membrane-initiated steroid signaling, we investigated the correlation of age in male chickens with the magnitude of responses to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)2D3 (130 pm) responsiveness as a function of age, showed a decreased intestinal Ca2+ transport. Western analyses of isolated basal lateral membranes indicated a decreased expression of the membrane-associated rapid response binding protein with increasing age. Saturation analyses of [3H]1,25-(OH)2D3 binding to basal lateral membranes, revealed an allosteric interaction identified as cooperative binding. A significant increase in Kd was observed with increasing age, indicating decreasing affinity. Determinations of the number of binding sites yielded a binding capacity of 190–250 fmol/mg protein during growth and maturation, whereas in adulthood (58 wk) saturable binding was no longer observed. Data obtained in parallel analys...
TL;DR: The results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.
Abstract: Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.
TL;DR: The unimolecular dsDNA microarray provides a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets and demonstrates that the nucleotides at different positions contribute differently to the p50p50/I g -κB binding interaction.