Showing papers on "Cooperativity published in 2008"
TL;DR: It is observed that at room temperature a fast exchange on the second time scale exists between molecules and stacks of sergeant (R)-1 and soldier 2, and that interconversion between M and P helices is fast at this temperature.
Abstract: On the basis of temperature-dependent UV-vis and circular dichroism (CD) spectroscopy measurements, we observed that C3-symmetrical discotic molecules, chiral (R)-1 and achiral 2, both self-assemble in a highly cooperative fashion. Chiral (R)-1 shows a higher degree of cooperativity, meaning it requires a larger nucleus before elongation sets in, as compared to achiral 2. Next to that, we investigated the mechanism of the "sergeants-and-soldiers" principle, where we found that the chiral sergeant (R)-1 strongly amplifies the preference in handedness of the mixed stacks of (R)-1 and 2. However, the elongation temperature and the degree of cooperativity are linearly dependent on both, at least in the regime above 4% of sergeant in the mixed system. Remarkably, we observed that at room temperature a fast exchange on the second time scale exists between molecules and stacks of sergeant (R)-1 and soldier 2, and that interconversion between M and P helices is fast at this temperature.
446 citations
01 May 2008
TL;DR: In this article, transient hyperglycemia was used to link NFκB-p65 gene expression with H3K4 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1).
Abstract: OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.
409 citations
TL;DR: Some of the manifestations of cooperativity that are most important in biology and drug discovery as they pertain to systems at different levels of complexity are discussed and aspects of this broadly important phenomenon that remain poorly understood are highlighted.
Abstract: Cooperative binding effects pervade biology. Only a few basic principles are at play, but in different biological contexts cooperativity appears in distinct guises to achieve different ends. Here I discuss some of the manifestations of cooperativity that are most important in biology and drug discovery as they pertain to systems at different levels of complexity and also highlight aspects of this broadly important phenomenon that remain poorly understood.
250 citations
TL;DR: Although the structure of the four‐octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4.
Abstract: Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.
237 citations
TL;DR: It is concluded that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in Terms of simpler mass‐action models.
Abstract: Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.
227 citations
TL;DR: Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization, and the relation between stimulus strength and receptor current shows positive cooperativity.
Abstract: Most vertebrate olfactory receptor neurons share a common G-protein–coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca 2+ ) through cyclic nucleotide-gated channels and a secondary efflux of Cl through Ca 2+ -gated Cl channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca 2+ . It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.
190 citations
TL;DR: The model accurately predicts the behavior of receptor mutants, such as the dimerization-defective Y246D-EGF receptor, which exhibit a single class of binding sites and provides a framework for understanding secondary dimer formation and lateral signaling in the EGF receptor family.
Abstract: Scatchard analysis of the binding of EGF to its receptor yields concave up plots that indicate the presence of two classes of binding sites. However, how two independent classes of sites arise from the expression of a single EGF receptor protein has never been adequately explained. Using a new analytical approach involving the simultaneous fitting of binding isotherms from cells expressing increasing levels of EGF receptors, we show that 125I-EGF-binding data can be completely explained by a model involving negative cooperativity in an aggregating system. This approach provides an experimentally determined value for the monomer–dimer equilibrium constant, which, for wild-type EGF receptors, corresponds to ≈50,000 receptors per cell. Therefore, changes in receptor expression within the physiological range can modulate the outcome of a signaling stimulus. Analysis of the L680N-EGF receptor mutant, in which the formation of asymmetric kinase domain dimers is blocked, indicates that the kinase dimers make a substantial energetic contribution to the ligand-independent association of EGF receptor monomers, but are not necessary for negative cooperativity. The model accurately predicts the behavior of receptor mutants, such as the dimerization-defective Y246D-EGF receptor, which exhibit a single class of binding sites and provides a framework for understanding secondary dimer formation and lateral signaling in the EGF receptor family.
175 citations
TL;DR: It is shown that positive allosteric cooperativity is generated by nucleotide and substrate binding during the transitions through the major conformational states: apo, intermediate, and closed.
Abstract: Allosteric signaling in proteins requires long-range communication mediated by highly conserved residues, often triggered by ligand binding. In this article, we map the allosteric network in the catalytic subunit of protein kinase A using NMR spectroscopy. We show that positive allosteric cooperativity is generated by nucleotide and substrate binding during the transitions through the major conformational states: apo, intermediate, and closed. The allosteric network is disrupted by a single site mutation (Y204A), which also decouples the cooperativity of ligand binding. Because protein kinase A is the prototype for the entire kinome, these findings may serve as a paradigm for describing long-range coupling in other protein kinases.
157 citations
TL;DR: Results indicate significant cooperativity between the halogen and hydrogen bonds in these complexes, much greater than that between hydrogen bonds.
Abstract: Ab initio calculations are used to provide information on H(3)N...XY...HF triads (X, Y=F, Cl, Br) each having a halogen bond and a hydrogen bond. The investigated triads include H(3)N...Br(2)-HF, H(3)N...Cl(2)...HF, H(3)N...BrCl...HF, H(3)N...BrF...HF, and H(3)N...ClF...HF. To understand the properties of the systems better, the corresponding dyads are also investigated. Molecular geometries, binding energies, and infrared spectra of monomers, dyads, and triads are studied at the MP2 level of theory with the 6-311++G(d,p) basis set. Because the primary aim of this study is to examine cooperative effects, particular attention is given to parameters such as cooperative energies, many-body interaction energies, and cooperativity factors. The cooperative energy ranges from -1.45 to -4.64 kcal mol(-1), the three-body interaction energy from -2.17 to -6.71 kcal mol(-1), and the cooperativity factor from 1.27 to 4.35. These results indicate significant cooperativity between the halogen and hydrogen bonds in these complexes. This cooperativity is much greater than that between hydrogen bonds. The effect of a halogen bond on a hydrogen bond is more pronounced than that of a hydrogen bond on a halogen bond.
151 citations
TL;DR: Here, the equilibrium states of the reactions in which n ligand bind a receptor sequentially, both non-cooperatively and in a cooperative fashion are studied, finding that n is not a good estimate, but only an upper bound, for the Hill coefficient.
Abstract: Hill functions follow from the equilibrium state of the reaction in which n ligands simultaneously
bind a single receptor. This result if often employed to interpret the Hill coefficient as
the number of ligand binding sites in all kinds of reaction schemes. Here, we study the equilibrium
states of the reactions in which n ligand bind a receptor sequentially, both non-cooperatively and
in a cooperative fashion. The main outcomes of such analysis are that: n is not a good estimate,
but only an upper bound, for the Hill coefficient; while the Hill coefficient depends quite strongly
on the cooperativity level among ligands. We finally use these results to discuss the feasibility
and constrains of using Hill functions to model the regulatory functions in mathematical models of
gene regulatory networks.
139 citations
TL;DR: It is suggested that a coupled equilibrium shift mechanism controls the efficient binding of CaM to a wide range of ligands.
TL;DR: Triazolophanes that incorporate pyridyl subunits in place of phenylenes show a heightened propensity to form 2:1 sandwich complexes with halides and Modest cooperative effects are observed for the snugly fitting F(-), Cl(-), and Br(-) halides while the too-large I(-) shows highly positive cooperativity.
Abstract: Triazolophanes that incorporate pyridyl subunits in place of phenylenes show a heightened propensity to form 2:1 sandwich complexes with halides Persistent iodide-based sandwiches are observed Binding constants confirm that the inward-facing electron pairs on the pyridyls destabilize the 1:1 complexes with halides The 1H NMR spectra verify that the sandwich complexes have two π-stacked triazolophanes rotated to allow registration between opposite dipoles on the pyridyls (directed inward) and triazoles (directed outward) These dipolar interactions cooperate to lower the pyridyl-based repulsions, therefore, increasing K2 Modest cooperative effects are observed for the snugly fitting F−, Cl−, and Br− halides while the too-large I− shows highly positive cooperativity
TL;DR: The relationship between open channel probability and phosphatidylglycerol content shows cooperativity, consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open.
TL;DR: The data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.
TL;DR: Understanding cooperativity provides insight into the mechanisms that govern assembly and disassembly of multicomponent complexes, and a significant challenge remains in tackling the thermodynamics and kinetics of the intermolecular interactions required for all cellular functions.
Abstract: The thermodynamic principle of cooperativity is used to drive the formation of specific macromolecular complexes during the assembly of a macromolecular machine Understanding cooperativity provides insight into the mechanisms that govern assembly and disassembly of multicomponent complexes Our understanding of assembly mechanisms is lagging considerably behind our understanding of the structure and function of these complexes A significant challenge remains in tackling the thermodynamics and kinetics of the intermolecular interactions required for all cellular functions
TL;DR: Results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
Abstract: The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
TL;DR: Application of the concept of an oligomeric allosteric enzyme to microsomal cytochromes P450 in combination with a general paradigm of multiple ligand occupancy of the active site provides an excellent explanation for complex manifestations of the atypical kinetic behavior of the enzyme.
Abstract: According to the initial hypothesis on the mechanisms of cooperativity in drug-metabolizing cytochromes P450, a loose fit of a single substrate molecule in the P450 active site results in a requirement for the binding of multiple ligand molecules for efficient catalysis. Although simultaneous occupancy of the active site by multiple ligands is now well established, there is increasing evidence that the mechanistic basis of cooperativity also involves an important ligand-induced conformational transition. Moreover, recent studies demonstrate that the conformational heterogeneity of the enzyme is stabilized by ligand-dependent interactions of several P450 molecules. Application of the concept of an oligomeric allosteric enzyme to microsomal cytochromes P450 in combination with a general paradigm of multiple ligand occupancy of the active site provides an excellent explanation for complex manifestations of the atypical kinetic behavior of the enzyme.
TL;DR: The results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PP PSP motifs represents a built-in amplifier for Wnt signaling by the L RP6 family of receptors.
TL;DR: Cooperativity is quantified and the computed positive cooperativity between cation-pi and hydrogen bonding interactions is rationalized through reduced variational space (RVS) and charge analyses.
Abstract: Quantum chemical calculations are performed to gauge the effect of cation−π and hydrogen bonding interactions on each other. M−phenol−acceptor (M = Li+ and Mg2+; acceptor = H2O, HCOOH, HCN, CH3OH, HCONH2 and NH3) is taken as a model ternary system that exhibits the cation−π and hydrogen bonding interactions. Cooperativity is quantified and the computed positive cooperativity between cation−π and hydrogen bonding interactions is rationalized through reduced variational space (RVS) and charge analyses.
TL;DR: The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches, and the techniques employed in the binding studies are categorized.
Abstract: P450s have attracted tremendous attention owing to not only their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely, the oxidation of unactivated C-H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s, including mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active-site features. Some of the substrate binding studies extending over a period of more than 40 years of dedicated work have been summarized in this review and categorized by the techniques employed in the binding studies.
TL;DR: Data indicate that both the cis monomer and the trans monomers and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.
TL;DR: Data from nucleotide analog interference mapping and mutagenesis support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P3a helix from both aptamers interface with the expression platform.
Abstract: The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.
TL;DR: ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady-state ATP hydrolysis, and it is proposed that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB.
TL;DR: These studies uniquely advance the understanding of both the energetics and kinetics of protein folding, and the recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings.
TL;DR: The theory unifies dynamic cooperativity and Hopfield-Ninio's kinetic proofreading mechanism for specificity amplification and shows Sigmoidal cooperative substrate binding to slowly fluctuating, monomeric enzymes is shown to arise from association pathways with very small probability but extremely long passage time.
TL;DR: The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.
Abstract: A crystal structure of the intact, dimeric phage λ cI repressor bound to a DNA operator site reveals an unusual architecture allowing the repressor to adopt a conformation that facilitates cooperative binding to adjacent operator sites. Bacteriophage λ has for many years been a model system for understanding mechanisms of gene regulation1. A ‘genetic switch’ enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the λ chromosome that are separated by about 2,400 base pairs (bp)2,3. A hallmark of the λ system is the pairwise cooperativity of repressor binding4. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact λ cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.
TL;DR: In this article, it was shown that the random arrival of diffusing signaling molecules at receptor sites constitutes a noise source that is not reduced by cooperativity, and that this noise source cannot be reduced by cooperation.
Abstract: Cooperative interactions in the binding of multiple signaling molecules is a common mechanism for enhancing the sensitivity of biological signaling systems. It is widely assumed this increase in sensitivity of the mean response implies the ability to detect smaller signals. Extending the classic work of Berg and Purcell [Biophys. J. 20, 193 (1977)] on the physical limits of chemoreception, we show that the random arrival of diffusing signaling molecules at receptor sites constitutes a noise source that is not reduced by cooperativity. Cooperativity makes reaching this limit easier, but cannot reduce the limit itself.
TL;DR: X-ray crystal structures of caspase-1 variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket for activity.
TL;DR: It is proposed that the oxygen-affinity of Hb is regulated by pentanary (the 5th-order time-dependent or dynamic) tertiary structural changes rather than the T-/R-quaternary structural transitions in Hb.
TL;DR: Proside, a method of in vitro selection, is employed to find stabilized variants of TEM-1 beta-lactamase from Escherichia coli and removes steric strain that originates from an overly tight packing of two helices in the wild-type protein.