Showing papers on "Cooperative binding published in 2007"

H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing

Abstract: H-NS is a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H-NS selectively silences specific genes of the bacterial chromosome. However, many studies have concluded that H-NS binds sequence-independently to DNA, leaving the molecular basis for its selectivity unexplained. We show that the negative regulatory element (NRE) of the supercoiling-sensitive Escherichia coliproU gene contains two identical high-affinity binding sites for H-NS. Cooperative binding of H-NS is abrogated by changes in DNA superhelical density and temperature. We further demonstrate that the high-affinity sites nucleate cooperative binding and establish a nucleoprotein structure required for silencing. Mutations in these sites result in loss of repression by H-NS. In this model, silencing at proU, and by inference at other genes directly regulated by H-NS, is tightly controlled by the cooperativity between bound H-NS molecules.

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

Abstract: G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.

Relating ligand binding to activation gating in CNGA2 channels

Abstract: Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 microM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

Structure-Function Studies of Allosteric Agonism at M2 Muscarinic Acetylcholine Receptors

Abstract: The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Tyr(177), and Thr(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the (172)EDGE(175) sequence had been neutralized and each Tyr(177) and Thr(423) was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr(177) and Thr(423), rather than the (172)EDGE(175) sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Resolving the fast kinetics of cooperative binding: Ca2+ buffering by calretinin.

Abstract: Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Nemethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-mus rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions ( approximately 100 nM) to approximately 1 muM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.

A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase

Abstract: The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod–Wyman–Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R–T equilibrium by using highly deuterated, 1H,13C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L′) between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is “invisible” in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R–T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R–T equilibrium, consistent with the Monod–Wyman–Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.

Molecular dynamics studies of AChBP with nicotine and carbamylcholine: the role of water in the binding pocket

Abstract: The acetylcholine-binding protein (AChBP) is homologous to the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) and other members of the Cys-loop family of neurotransmitter receptors. The high-resolution X-ray structures of AChBP mean it has been used as a model from which to understand agonist and antagonist binding to nAChRs. We present here a molecular dynamics (MD) study of AChBP with nicotine and carbamylcholine bound. Our results suggest that the ligand imposes rigidity on the binding pocket residues. The simulations also suggest that the protein undergoes breathing motions with respect to the five-fold axis, a motion that has been postulated to be related to gating in the nAChR. We analyzed the behaviour of the water molecules in and around the binding site and found that they occupied five distinct sites within the binding pocket. Water occupied these sites in the absence of ligand, but the presence of ligand increased the probability that a water molecule would be found in these sites. Finally, we demonstrate how the positions of these waters might be used in the design of new ligands by comparing the positions of these sites with other recent structures.

A Computational Investigation of Allostery in the Catabolite Activator Protein

Abstract: The catabolite activator protein is a dimer that consists of two cAMP-binding subunits, each containing a C-terminus DNA-binding module and a N-terminus ligand binding domain. The system is well-known to exhibit negative cooperativity, whereby the binding of one cAMP molecule reduces the binding affinity of the other cAMP molecule by 2 orders of magnitude, despite the large separation between the cAMP binding pockets. Here we use extensive explicit-solvent molecular dynamics simulations (135 ns) to investigate the allosteric mechanism of CAP. Six trajectories were carried out for apo, singly liganded, and doubly liganded CAP, both in the presence and absence of DNA. Thorough analyses of the dynamics through the construction of dynamical cross-correlated maps, as well as essential dynamics analyses, indicated that the system experienced a switch in motion as a result of cAMP binding, in accordance with recent NMR experiments carried out on a truncated form of the protein. Analyses of conformer structures collected from the simulations revealed a remarkable event: the DNA-binding module was found to dissociate from the N-terminus ligand binding domain. An interesting aspect of this structural change is that it only occurred in unoccupied subunits, suggesting that the binding of cAMP provides additional stability to the system, consistent with the increase in entropy that was observed in our calculations and from isothermal titration calorimetry. Analysis of the distribution of intrinsic disorder propensities in CAP amino acid sequence using PONDR VLXT and VSL1 predictors revealed that the region connecting ligand-binding and DNA-binding domains of CAP have the potential to exhibit increased flexibility. We complemented these trajectories with free energy calculations following the MM-PBSA approach on more than 2000 snapshots that included 880 normal mode analysis. The resulting free energy differences between the singly liganded and doubly liganded states were in excellent agreement with isothermal titration calorimetry data. When the free energy calculations were carried out in the presence of DNA, we discovered that a switch in cooperativity occurred, so that the binding of the first cAMP promoted the binding of the other cAMP. The components of the free energy reveal that this effect is mainly entropic in nature, whereby the DNA reduces the degree of tightening that is observed in its absence, thereby promoting binding of the second cAMP. This finding prompted us to propose a new mechanism by which CAP triggers the transcription activation that is based on an order to disorder transition mediated by cAMP binding as well as DNA.

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble.

Abstract: Conformational fluctuations in proteins have emerged as a potentially important aspect of biological function, although the precise relationship and the implications have yet to be fully explored. Numerous studies have reported that the binding of ligand can influence fluctuations. However, the role of the binding site in mediating these fluctuations is not known. Of particular interest is whether in addition to serving as structural scaffolds for recognition and catalysis, active-site residues may also play a role in modulating the cooperative network. To address this question, we employ an experimentally validated ensemble-based description of proteins to elucidate the extent to which perturbations at different sites can influence the cooperative network in the protein. Applying this method to a database of test proteins, it is found statistically that binding sites are located in regions most able to affect the cooperative network, even for cooperative interactions between residues distant to the binding sites. This indicates that the conformational manifold under native conditions is determined by the network of cooperative interactions within the protein and suggests that proteins have evolved to use these conformational fluctuations in carrying out their functions. Furthermore, because the energetic coupling pattern calculated for each protein is robust and relatively insensitive to sequence, these studies further suggest that binding sites evolved in regions of the protein that are inherently poised to take advantage of the fluctuations in the native structure.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

Abstract: Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. British Journal of Pharmacology (2007) 151, 84–95. doi:10.1038/sj.bjp.0707196

Phenyl-calix[4]arene-Based Fluorescent Sensors: Cooperative Binding for Carboxylates

Abstract: Tetrakis-(4-carbamoylphenyl)-substituted and tetrakis-(4-amidophenyl)-substituted calix[4]arenes as well as the monomeric biphenylcarbamate have been synthesized as fluorescent receptors for anion sensing. Their binding properties with various anions including F-, CH3COO-, Ph-COO-, and H2PO4- were investigated by fluorescence titrations, Job plot experiments, 1H NMR spectroscopies, and ESI-MS measurements. Importantly, we have found that calix[4]arene-based sensors exhibit greatly enhanced binding affinity and selectivity toward carboxylates. The binding associations of tetrakis-(4-carbamoylphenyl)-substituted calix[4]arene for carboxylates are 1-2 orders of magnitude greater than those of the monomeric biphenylcarbamate sensor. Such an enhancement in the binding affinity and selectivity is attributed to the cooperative binding of the multiple ligating groups as revealed from the ab inito DFT calculations. Although tetrakis-(4-amidophenyl)-substituted calix[4]arene exhibited relatively weaker binding affinity toward anions, its superior binding selectivity for acetate ion over fluoride ion is evident. Our results also suggest that carbamate functionality is a useful H-bond donor for hydrogen-bonding interactions in molecular recognition and supramolecular chemistry.

Thermodynamics of the interaction between O-acetylserine sulfhydrylase and the C-terminus of serine acetyltransferase.

Abstract: Cysteine biosynthesis in plants is partly regulated by the physical association of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). Interaction of OASS and SAT requires only the 10 C-terminal residues of SAT. Here we analyze the thermodynamics of formation of a complex of Arabidopsis thaliana OASS (AtOASS) and the C-terminal ligand of AtSAT (C10 peptide) as a function of temperature and salt concentration using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Our results suggest that the C-terminus of AtSAT provides the major contribution to the total binding energy in the plant cysteine synthase complex. The C10 peptide binds to the AtOASS homodimer in a 2:1 complex. Interaction between AtOASS and the C10 peptide is tight (Kd = 5−100 nM) over a range of temperatures (10−35 °C) and NaCl concentrations (0.02−1.3 M). AtOASS binding of the C10 peptide displays negative cooperativity at higher temperatures. ITC studies reveal compensating changes in the enthalpy and...

Tuning the strength and selectivity of ion-pair recognition using heteroditopic calix[4]arene-based receptors

Abstract: The syntheses, characterisation and ion binding properties of two novel heteroditopic calix[4]arene receptors are reported. These systems were found to demonstrate cooperative binding of certain ion-pairs, with the selectivity depending critically on the structure of the receptors. Furthermore, a macrocyclic effect for ion-pair recognition was observed to operate.

Structural dynamics of the cooperative binding of organic molecules in the human cytochrome P450 3A4.

Abstract: Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C−H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in si...

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Abstract: Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the low-activity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, low-affinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.

A cationic host displaying positive cooperativity in water.

Abstract: The affinity of guanidinium and Cu(II) containing hosts for polycarboxylate guests is studied in water by using UV-visible spectroscopy and isothermal titration calorimetry. By combining a Cu(II) coordination site and a diguanidinium moiety around a single scaffold it is found that the Gibbs free-energy release upon binding is greater than could be expected based on the sum of the free energy released by the binding functional groups when operating in isolation. This effect is known as positive cooperativity and has rarely been observed with synthetic hosts in water. The isothermal titration calorimetry data suggest that the source of this positive cooperativity is enthalpic in origin. We propose that covalently positioning the cationic binding moieties in close proximity destabilizes the unbound host, and thereby the stabilizing effect of binding the anionic carboxylates is exothermic and also, the source of positive cooperativity observed herein.

Definition of an electrostatic relay switch critical for the cAMP‐dependent activation of protein kinase A as revealed by the D170A mutant of RIα

Abstract: The Regulatory (R) subunit of Protein Kinase A (PKA) inhibits its kinase activity by shielding the Catalytic (C) subunit from physiological substrates. This inhibition is reversed in response to extra-cellular signals that increase cAMP levels in the cytoplasm. Upon cAMP binding to R, C is allosterically released from R, activating a spectrum of downstream signaling cascades. Crystallographic data indicated that a series of distinct conformational changes within CBD-A must occur to relay the cAMP signal from the cAMP binding site to the R:C interaction interface. One critical cAMP relay site within the CBD-A of R has been identified as Asp170 because the D170A mutation selectively reduces the negative cooperativity between the cAMP- and C-recognition sites (i.e. the KD for the R:C complex in the presence of cAMP is reduced by more than 12-fold), without significantly compromising the high affinity of R for both binding partners. Here, utilizing an integrated set of comparative NMR analyses we have elucidated how this critical electrostatic switch is able to control the interaction network which transmits the cAMP signal within CBD-A. The D170A-induced variations in backbone chemical shifts as well as in hydrogen-deuterium and hydrogen-hydrogen exchange profiles show that Asp170 not only plays a pivotal role in controlling the local conformation of the phosphate binding cassette (PBC), where cAMP docks, but also significantly affects the long-range cAMP-dependent interaction network that extends from the PBC to the three major sites of C-recognition. We also found that the D170A mutation promotes partial unfolding, thus assisting the uncoupling of the alpha- and beta-subdomains of CBD-A as required for the major alpha-helical conformational re-arrangement necessary for C-binding. Overall, the emerging map of allosteric networks features Asp170 as an essential component of an electrostatic switch mechanism that stabilizes the conformation of the PBC region for optimal interaction with cAMP and that is also crucial for relaying allosteric effects leading to C subunit activation. Taken together, our results consolidate the interdependence between the Asp170 relay site and the R:C interaction interface. Furthermore, they provide insight into the driving forces for the in vivo formation of intermediate PKA ternary complexes. Finally, our current study is relevant for elucidating the antagonistic properties of Rp-cAMPS on PKA by providing a detailed picture of the long-range effects of the altered interaction between this analog and the PBC.