Showing papers on "Cooperativity published in 1996"

Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.

Abstract: In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a Michaelis-Menten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.

Single-domain/bound calcium hypothesis of transmitter release and facilitation.

Abstract: 1. We describe a model of transmitter release that is based on the finding that release can be gated during the opening of individual Ca2+ channels, suggesting that the release site can be activated by the Ca2+ domain under a single channel. In this model each release site contains four independent Ca2+ binding sites or gates with unbinding kinetics graded from slow to fast and affinities ranging from high to low. All four gates must be bound for release to occur. Thus synaptic dynamics are governed by the kinetics of Ca2+ binding and unbinding from release sites, not Ca2+ diffusion. 2. Fast facilitation occurs when an action potential invades a terminal with one or more ions remaining bound to the release sites. Residual free Ca2+ is not necessary for facilitation with this mechanism, but if present it would enhance facilitation by binding to high-affinity gates between pulses. 3. This model can account for key features of release. These include fourth-power cooperativity with regard to external Ca2+; a release time course that is virtually independent of an increase in quantal content; an inverse relation between external Ca2+ and the degree of facilitation; and a steplike increase in facilitation with increasing stimulus frequency, with each step corresponding to a unitary decline in the Ca2+ cooperativity. 4. Facilitation of single-channel-based secretion is shown to be robust even if channel opening is stochastic. Spontaneous release of transmitter, assumed to be due in part to spontaneous Ca2+ channel openings, is shown to be elevated during and after a train of impulses. 5. An extension of the model to include multiple Ca2+ channels per release site demonstrates that one role of overlapping Ca2+ domains may be to accentuate depolarization-evoked release relative to spontaneous release.

Characteristic length of the glass transition

Abstract: The definition of molecular cooperativity is discussed. The characteristic length of the glass transition describes the size of this cooperativity. Differential scanning calorimetry (DSC) and heat capacity spectroscopy (HCS) results of a series of poly(n-alkyl methacrylates) (alkyl = methyl, ethyl, propyl, butyl, pentyl, hexyl, and octyl) and a series of statistical copolymers poly(n-butylmethacrylate-stat-styrene) are discussed in terms of molecular cooperativity in the αβ splitting region, where a high-frequency dispersion zone a splits off into the main transition zone α and a Goldstein Johari process β at lower frequencies. The characteristic length tends to small values of order one monomer diameter in the splitting region for scenarios with an α relaxation onset. The statements about the size scale of cooperativity are conditional upon certain assumptions leading to the equation used for calculation of this size from HCS and DSC data. The step height of heat capacity (Δcp) and, with less certainty, the square root of the cooperativity volume or number (V1/2α or 1/2α) are proportional to the temperature distance from the cooperativity onset, T = Tons. © 1996 John Wiley & Sons, Inc.

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor

Abstract: The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.

Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin

Abstract: ↵ Present address: Department of Biochemistry and Molecular Biology, Rice University, Houston, TX 77005.

Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage.

Abstract: Structural and biochemical studies of Oct-1 POU domain-DNA interactions have raised important questions about cooperativity and the role of the linker connecting the POU-specific domain and the POU homeo domain. To analyze these interactions, we have studied binding of the isolated domains. Surprisingly, we find that two unlinked polypeptides corresponding to the POU-specific domain and the POU homeo domain bind cooperatively to the octamer site and have a coupling energy of 1.6 kcal/mole. We suggest that overlapping DNA contacts near the center of the octamer site may be the source of this cooperativity, as there are no protein-protein contacts between the domains in the crystal structure of the Oct-1 POU domain-DNA complex. These studies also have allowed us to describe the thermodynamic contribution of the linker (present in the intact POU domain) in terms of an effective concentration (3.6 mM). The broader implications for understanding cooperativity in protein-DNA recognition and gene regulation are discussed.

A kinetic mechanism for nicotinic acetylcholine receptors based on multiple allosteric transitions

Abstract: Nicotinic acetylcholine receptors are transmembrane oligomeric proteins that mediate interconversions between open and closed channel states under the control of neurotransmitters. Fast in vitro chemical kinetics and in vivo electrophysiological recordings are consistent with the following multi-step scheme. Upon binding of agonists, receptor molecules in the closed but activatable resting state (the Basal state, B) undergo rapid transitions to states of higher affinities with either open channels (the Active state, A) or closed channels (the initial Inactivatable and fully Desensitized states, I and D). In order to represent the functional properties of such receptors, we have developed a kinetic model that links conformational interconversion rates to agonist binding and extends the general principles of the Monod-Wyman-Changeux model of allosteric transitions. The crucial assumption is that the linkage is controlled by the position of the interconversion transition states on a hypothetical linear reaction coordinate. Application of the model to the peripheral nicotine acetylcholine receptor (nAChR) accounts for the main properties of ligand-gating, including single-channel events, and several new relationships are predicted. Kinetic simulations reveal errors inherent in using the dose-response analysis, but justify its application under defined conditions. The model predicts that (in order to overcome the intrinsic stability of the B state and to produce the appropriate cooperativity) channel activation is driven by an A state with a Kd in the 50 nM range, hence some 140-fold stronger than the apparent affinity of the open state deduced previously. According to the model, recovery from the desensitized states may occur via rapid transit through the A state with minimal channel opening, thus without necessarily undergoing a distinct recovery pathway, as assumed in the standard 'cycle' model. Transitions to the desensitized states by low concentration 'pre-pulses' are predicted to occur without significant channel opening, but equilibrium values of IC50 can be obtained only with long pre-pulse times. Predictions are also made concerning allosteric effectors and their possible role in coincidence detection. In terms of future developments, the analysis presented here provides a physical basis for constructing more biologically realistic models of synaptic modulation that may be applied to artificial neural networks.

Interaction of CnTAB with Sodium (Carboxymethyl)cellulose: Effect of Polyion Linear Charge Density on Binding Isotherms and Surfactant Aggregation Number

Abstract: The interaction of alkyltrimethylammonium bromide (CnTAB) with sodium (carboxymethyl)cellulose (NaCMC) in dilute aqueous solutions is investigated. The effect of salt on the binding to NaCMC of high linear charge density (ξ = 3.0) is investigated using a surfactant-sensitive electrode and compared with the behavior in other polyelectrolyte systems. From time-resolved fluorescence quenching experiments the aggregation numbers for C12TAB and C14TAB micelles formed with NaCMC as counterion (no salt) are obtained as 58 and 83, respectively, i.e., close to the values for the corresponding polyelectrolyte free systems. The binding of C12TAB to NaCMC with different linear charge densities (ξ = 0.8, 1.5, 2.0, 2.3, 3.0) is determined in the presence of 2.5 mM sodium bromide. From the binding isotherms, the critical aggregation concentration (cac) of the surfactant and the cooperativity in the binding are estimated. The cac decreases and the cooperativity increases with increasing charge density of the polyelectrol...

Structural Determinants within Pbx1 That Mediate Cooperative DNA Binding with Pentapeptide-Containing Hox Proteins: Proposal for a Model of a Pbx1-Hox-DNA Complex

Abstract: Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.

Cooperative effects in water trimers. The performance of density functional approaches

Abstract: The performance of B-LYP, B3-LYP, B3-P86 and B3-PW91 density functional to describe cooperative effects in water trimer was investigated. The geometries and vibrational frequencies of the monomer, the dimer and the trimer were obtained using a 6-31+G(d,p) basis set, while the final energies were calculated at the 6-311++G(3df,2p) level. Our results show that B3-LYP and B-LYP functionals seem to be a good alternative to ab initio calculations to account for cooperative or non-pairwise effects, either if these effects are measured in terms of additive interaction energies or in terms of the so-called cooperativity factors.

Synthesis of a Stereoblock Polyketone through Ancillary Ligand Exchange

Abstract: We report a new strategy for synthesis of stereoblock polymers involving ancillary ligand exchange during a living transition-metal-catalyzed polymerization. We have also demonstrated cooperativity between chain-end and site control mechanisms through ligand exchange. On the basis of these results, stereoblock copolymer synthesis was approached by first growing an isotactic segment with I-2, then switching on syndiotactic growth by displacing the bisoxazoline ligand from the Pd(II) catalyst with bipyridine. 18 refs., 1 fig.

On the Origin of the Cooperativity of Protein Folding: Implications From Model Simulations

Abstract: There is considerable experimental evidence that the cooperativity of protein folding resides in the transition from the molten globule to the native state. The objective of this study is to examine whether simplified models can reproduce this cooperativity and if so, to identify its origin. In particular, the thermodynamics of the conformational transition of a previously designed sequence (A. Kolinski, W. Galazka, and J. Skolnick, J. Chem. Phys. 103: 10286-10297, 1995), which adopts a very stable Greek-key beta-barrel fold has been investigated using the entropy Monte Carlo sampling (ESMC) technique of Hao and Scheraga (M.-H. Hao and H.A. Scheraga, J. Phys. Chem. 98: 9882-9883, 1994). Here, in addition to the original potential, which includes one body and pair interactions between side chains, the force field has been supplemented by two types of multi-body potentials describing side chain interactions. These potentials facilitate the protein-like pattern of side chain packing and consequently increase the cooperativity of the folding process. Those models that include an explicit cooperative side chain packing term exhibit a well-defined all-or-none transition from a denatured, random coil state to a high-density, well-defined, nativelike low-energy state. By contrast, models lacking such a term exhibit a conformational transition that is essentially continuous. Finally, an examination of the conformations at the free-energy barrier between the native and denatured states reveals that they contain a substantial amount of native-state secondary structure, about 50% of the native contacts, and have an average root mean square radius of gyration that is about 15% larger than native.

Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme.

Abstract: Amino-terminal and carboxy-terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452). The wild-type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L-Phe) binding (Hill coefficient h= 2.2, S0.5= 154 μM). The deletion mutants lacking the carboxy-terminal 24 amino acids hPAH(Ser2-Gln428) and hPAH(Gly103-Gln428) formed catalytically active dimers, and incubation with L-Phe did not promote the formation of tetramers, a characteristic property of dimeric wt-hPAH. The carboxy-terminus thus seems to contain a motif required for dimer-dimer interaction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Ser2-Gln428) and hPAH(Glyl03-Gln428) were all activated by prior incubation with L-Phe, but did not reveal any positive cooperativity of substrate binding (h= 1.0). The activation by L-Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino-terminal sequence, i.e. wt-hPAH and the Ser2 - Gln428 mutant. The amino-terminal deletion mutants hPAH(Asp112–Lys452) and hPAH(Gly103-Gln428) revealed high specific activity, increased apparent affinity for L-Phe (S0.5= 60 μM) and a tryptophan fluorescence emission spectrum similar to that of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt-hPAH amino-terminal sequence. Our results are compatible with a model in which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino-terminal domain to the high-affinity binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.

NMR Studies of Cooperativity in the Tetrahaem Cytochrome c3 from Desulfovibrio vulgaris

Abstract: The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e−/e−) and heterotropic (e−/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVc3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.

Mechanism of differential activities of ofloxacin enantiomers.

Abstract: Ofloxacin, a potent quinolone antibacterial agent, has a tricyclic ring structure with a methyl group attached to the asymmetric carbon at the C-3 position on the oxazine ring. The S isomer (DR-3355) of ofloxacin has antibacterial activity up to 2 orders of magnitude greater than that of the R isomer (DR-3354). This differential antibacterial activity was not due to different drug transport mechanisms of the two isomers but was found to be derived from the inhibitory activity against the target enzyme, DNA gyrase. Previous mechanistic studies have suggested that the bactericidal effect of the drug is mediated through the stabilization of a cleavable complex via a cooperative drug binding process to a partially denatured DNA pocket created by DNA gyrase. The drug binds to supercoiled DNA in a manner similar to that to which it binds to the enzyme-DNA complex. In the present studies, we first examined the binding of the two radiolabeled ofloxacin enantiomers to supercoiled pUC9 plasmid DNA. Surprisingly, the two enantiomers possessed similar apparent binding affinities and binding cooperatives. The major difference in binding between the two stereoisomers was the molar binding ratio: 4 for the more active S isomer versus 2 for the less active R isomer. We next examined the relative binding potencies of the stereoisomers to the DNA-DNA gyrase complex. The results of a competition assay showed that (S)-ofloxacin binds 12-fold better to the complex than (R)-ofloxacin. The binding potencies of the two enantiomers and two other quinolones correlated well with their respective concentrations causing 50% inhibition against DNA gyrase. The results are interpreted by a stacking model by using the concept of the cooperative drug-DNA binding mechanism, indicating that the potencies of quinolones cannot be determined solely by the DNA binding affinity and cooperativity but can also be determined by their capability in maximally saturating the binding site. The capability of the drug in saturating the binding pocket manifests itself in an increased efficacy at inhibiting the enzyme through a direct interaction between the drug and the enzyme. The results augment the previous suggestion that the binding pocket in the enzyme-DNA complex involves multiple receptor groups including not only DNA bases but also a gyrase subunit. The higher level of potency of (S)-ofloxacin is proposed to derive from the fact that a greater number of molecules are assembled in the pocket. This greater number of molecules optimizes the interaction between the drug and the enzyme, possibly through a contact between the C-7 substituent and the quinolone pocket on the B subunit of DNA gyrase.

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Abstract: Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

IL-10 cooperates with TNF-alpha to activate HIV-1 from latently and acutely infected cells of monocyte/macrophage lineage.

Abstract: IL-10 is elevated in HIV-1-infected individuals and has been implicated in disease progression. In this study, we investigated the effects of IL-10 on the activation of HIV-1 from infected monocytes and macrophages. Although IL-10 alone did not induce HIV-1 replication, in the presence of TNF-alpha, IL-10 markedly enhanced virion production from a chronically infected promonocytic cell line (U1) and in acutely infected monocyte-derived macrophages. Neutralizing mAbs to IL-10 and TNF-alpha indicated that both cytokines were essential for the induction and were required to generate a synergistic increase in virus expression. The effects of the two cytokines were distinguishable functionally since pretreatment with TNF-alpha attenuated the cytokine cooperativity, while pretreatment with IL-10 potentiated their cooperativity, suggesting that IL-10 and TNF-alpha play different roles in the activation of virus. Northern blot analysis as well as Ab blocking and cytokine secretion studies indicated that the induction of either endogenous TNF-alpha or IL-10 was not involved in the cooperativity, nor was an up-regulation of TNF-alpha receptors. In combination with TNF-alpha, IL-10 stimulated activating protein-1 (AP-1) and nuclear factor (NF)-kappa B binding activities and cooperated to increase HIV-1 steady-state mRNA levels and enhance long terminal repeat-directed transcription through activation of the NF-kappa B binding sites, suggesting the IL-10 effect occurs at least in part at the transcriptional level. These results indicate that IL-10, in addition to down-regulating the cellular immune response to HIV-1, may also play a role in TNF-alpha-mediated activation of HIV-1 replication in the monocyte/macrophage lineage.

Structural basis for the root effect in haemoglobin.

Abstract: The remarkable ability of Root effect haemoglobins to pump oxygen against high O2 gradients results from extreme, acid-induced reductions in O2 affinity and cooperativity. The long-sought mechanism for the Root effect, revealed by the 2 A crystal structure of the ligand-bound haemoglobin from Leiostomus xanthurus at pH 7.5, unexpectedly involves modulation of the R-state. Key residues strategically assemble positive-charge clusters across the allosteric β1β2-interface in the R-state. At low βH, protonation of the βN terminus and His 147(HC3)β within these clusters is postulated to destabilize the R-state and promote the acid-triggered, allosteric R→T switch with concomitant O2 release. Surprisingly, a set of residues specific to Root effect haemoglobins recruit additional residues, conserved among most haemoglobins, to produce the Root effect.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo

Abstract: Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.