Showing papers on "Cooperativity published in 2001"

Positive allosteric systems designed on dynamic supramolecular scaffolds: toward switching and amplification of guest affinity and selectivity.

Abstract: Positive homotropic allosterism appears in important information transduction processes where chemical and physical signals are efficiently amplified. The phenomena are ubiquitous in nature, but the general methodology for the design of such allosteric systems is not yet established in an artificial system. This account reviews such artificial receptors that can bind guest ions and molecules in a positive allosteric manner and discusses what kinds of factors are indispensable as scaffolds in the design of this novel class of allosteric systems and what common factors are needed to realize the cooperativity. It has been shown that the scaffolds are mostly dynamic and are skillfully combined with the molecular recognition systems so that the subsequent guest binding can occur more favorably than the first guest binding. In addition, it has been suggested that positive homotropic allosterism can be utilized as a new strategy to attain high guest selectivity and guest affinity which cannot be attained by conv...

Coordination sphere flexibility of active-site models for Fe-only hydrogenase: studies in intra- and intermolecular diatomic ligand exchange.

Abstract: A series of dinuclear complexes, (μ-SRS)Fe2(CO)6 (R = −CH2CH2−, −CH2CH2CH2−, −CH2−C6H4−CH2−; edt, pdt, and o-xyldt, respectively) has been examined for specific characteristics that might relate to structural similarity with the active site of Fe-only hydrogenases. Variable-temperature proton NMR studies display the fluxionality of the iron−dithiocyclohexane unit in (μ-pdt)Fe2(CO)6 while in the (μ-o-xyldt)Fe2(CO)6 compound, the bridge is fixed. Temperature-dependent 13C NMR spectral studies establish intramolecular CO site exchange localized on discrete Fe(CO)3 units in all complexes, which is influenced by steric effects of the μ-SRS unit. Kinetic studies of intermolecular CO/CN- ligand-exchange reactions establish associative or Ia mechanisms in sequential steps to form the dicyano dianion, (μ-SRS)[Fe(CO)2(CN)]2= with 100% selectivity. Theoretical calculations (DFT) of transition states in the intramolecular site-exchange processes lead to a rationale for the interesting cooperativity in the CN-/CO inte...

Chiral Recognition in Bis-Urea-Based Aggregates and Organogels through Cooperative Interactions.

Abstract: Chiral recognition in organogels occurs in the coassembly of the chiral gelator 1 with the chiral guest molecule 2. The diastereomeric aggregates formed from (R,R)-2 with (R,R)-1 or (S,S)-1 were found to be truly different in structure and strength. Cooperativity makes a major contribution to the chiral recognition in this system.

Identifying target sites for cooperatively binding factors

Abstract: Motivation: Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. Though several methods exist for identification of individual protein binding site patterns in DNA sequences, there are few methods for discovery of binding site patterns for cooperatively acting factors. Here we present an algorithm, Co-Bind (for COperative BINDing), for discovering DNA target sites for cooperatively acting transcription factors. The method utilizes a Gibbs sampling strategy to model the cooperativity between two transcription factors and defines position weight matrices for the binding sites. Sequences from both the training set and the entire genome are taken into account, in order to discriminate against commonly occurring patterns in the genome, and produce patterns which are significant only in the training set. Results: We have tested Co-Bind on semi-synthetic and real data sets to show it can efficiently identify DNA target site patterns for cooperatively binding transcription factors. In cases where binding site patterns are weak and cannot be identified by other available methods, Co-Bind, by virtue of modeling the cooperativity between factors, can identify those sites efficiently. Though developed to model protein‐ DNA interactions, the scope of Co-Bind may be extended to combinatorial, sequence specific, interactions in other macromolecules. Availability: The program is available upon request from the authors or may be downloaded from http://ural.wustl. edu.

Selective recognition of an alkali halide contact ion-pair.

Abstract: For more than 30 years there has been an active and continued effort to develop synthetic receptors for anions and cations in organic solvents.1 We2 and others3 have shown that if both of the counterions in a target salt have localized charges then the consequent ion-pairing of the salt can dramatically lower receptor/ ion binding affinities and alter binding selectivities. One way to counter this problem is to develop ditopic receptors that can simultaneously bind both of the counterions.4 Recently, we investigated the salt binding properties of receptor 1 and found that the presence of 1 molar equiv of Na+ or K+ ion increases the 1/Classociation constant by slightly less than ten.5 An X-ray crystal structure showed that receptor 1 binds NaCl as a solvent separated ion-pair. We felt that the binding cooperativity would be improved if the salt were bound to the receptor as a contact ion-pair. Thus, we designed macrobicyclic receptor 2 as a saltbinding analogue of 1 but with a smaller distance between the anion and cation binding sites.6 Using NMR spectroscopy and X-ray crystallography we find that 2 is the first example of a ditopic salt-receptor that binds a contact ion-pair in solution more strongly than either of the free ions.

Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching.

Abstract: The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid chaperone that facilitates the rearrangement of nucleic acids into conformations containing the maximum number of complementary base pairs. We use an optical tweezers instrument to stretch single DNA molecules from the helix to coil state at room temperature in the presence of NC and a mutant form (SSHS NC) that lacks the two zinc finger structures present in NC. Although both NC and SSHS NC facilitate annealing of complementary strands through electrostatic attraction, only NC destabilizes the helical form of DNA and reduces the cooperativity of the helix-coil transition. In particular, we find that the helix-coil transition free energy at room temperature is significantly reduced in the presence of NC. Thus, upon NC binding, it is likely that thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations to find the lowest energy state. The reduced cooperativity allows these fluctuations to occur in the middle of complex double-stranded structures. The reduced stability and cooperativity, coupled with the electrostatic attraction generated by the high charge density of NC, is responsible for the nucleic acid chaperone activity of this protein.

Multisite Kinetic Models for CYP3A4: Simultaneous Activation and Inhibition of Diazepam and Testosterone Metabolism

Abstract: Some substrates of cytochrome P450 (CYP) 3A4, the most abundant CYP in the human liver responsible for the metabolism of many structurally diverse therapeutic agents, do not obey classical Michaelis-Menten kinetics and demonstrate homotropic and/or heterotropic cooperativity. The unusual kinetics and differential effects observed between substrates of this enzyme confound the prediction of drug clearance and drug-drug interactions from in vitro data. We have investigated the hypothesis that CYP3A4 may bind multiple molecules simultaneously using diazepam (DZ) and testosterone (TS). Both substrates showed sigmoidal kinetics in B-lymphoblastoid microsomes containing a recombinant human CYP3A4 and reductase. When analyzed in combination, TS activated the formation of 3-hydroxydiazepam (3HDZ) and N -desmethyldiazepam (NDZ) (maximal activation 374 and 205%, respectively). For 3HDZ, V max values remained constant with increasing TS, whereas the S 50 and Hill values decreased, tending to make the data less sigmoidal. Similar trends were observed for the NDZ pathway. DZ inhibited the formation 6β-hydroxytestosterone (maximal inhibition, 45% of control), causing a decrease in V max but no significant change to the S 50 and Hill values, suggesting that DZ may inhibit via a separate effector site. Multisite rate equation models have been derived to explore the analysis of such complex kinetic data and to allow accurate determination of the kinetic parameters for activation and inhibition. The data and models presented are consistent with proposals that CYP3A4 can bind and metabolize multiple substrate molecules simultaneously; they also provide a generic solution for the interpretation of the complex kinetic data derived from CYP3A4 substrates.

Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions.

Abstract: Cytochromes P450 (CYP) 3A4 is the most abundant human hepatic CYP isoform catalyzing the metabolism of approximately 50% of therapeutic agents. In addition to inhibition or induction, CYP3A4 is subject to stimulation, termed homotropic (substrate stimulation) and heterotropic (stimulation by effectors) cooperativity. The heterotropic cooperativity of CYP3A4 may result from an increase in Vmax, a decrease in Km or a combination of the two and sometimes exhibits regio-selectivity when the enzyme is involved in two or more metabolic pathways for a single substrate. An effector of CYP3A4 can also be a substrate; its metabolism may or may not be inhibited by another substrate. These characteristics of heterotropic cooperativity of CYP3A4 have been interpreted in the context of two binding domains in the active site of the enzyme, two substrate binding plus a distinct allosteric binding site, multiple enzyme conformations or multiple binding sites accompanied by conformational changes. Examples of in vivo CYP cooperativity are rare; representative cases include flavone-dependent stimulation of zoxazolamine metabolism in rats and enhancement of CYP3A-mediated hepatic clearance of diclofenac by quinidine in monkeys. Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxylation of warfarin in human hepatocyte systems. These data imply that CYP cooperativity has the potential to cause in vivo drug-drug interactions. Because cooperative and inhibitory responses from CYP3A4 are known to be substrate-dependent, projection of the pharmacokinetics of an investigational drug and CYP-associated risks of drug-drug interactions in humans can be very complex. Further investigation of CYP cooperativity is warranted.

Cooperativity in Drug−DNA Recognition: A Molecular Dynamics Study

Abstract: NMR studies have shown that the minor groove-binding ligand Hoechst 33258 binds to the two T4/A4 tracts within the duplex d(CTTTTCGAAAAG)2 in a highly cooperative manner, such that in titration experiments no intermediate 1:1 complex can be detected. The NMR-derived structures of the free DNA and the 2:1 complex have been obtained, but can shed little light on what the origins of this cooperativity may be. Here we present the results of a series of molecular dynamics simulations on the free DNA, the 1:1 complex, and the 2:1 complex, which have been designed to enable us to calculate thermodynamic parameters associated with the molecular recognition events. The results of the molecular dynamics studies confirm that structural factors alone cannot explain the cooperativity observed, indeed when enthalpic and hydration factors are looked at in isolation, the recognition process is predicted to be slightly anticooperative. However, when changes in configurational entropy are taken into account as well, the ov...

Dioxygen binding to deoxyhemocyanin: electronic structure and mechanism of the spin-forbidden two-electron reduction of o(2).

Abstract: Spectroscopically calibrated DFT is used to investigate the reaction coordinate of O(2) binding to Hemocyanin (Hc). A reaction path is calculated in which O(2) approaches the binuclear copper site with increasing metal-ligand overlap, which switches the coordination mode from end-on eta(1)-eta(1), to mu-eta(1):eta(2), then to butterfly, and finally to the planar [Cu(2)(mu-eta(2):eta(2)O(2))] structure. Analysis of the electronic structures during O(2) binding reveals that simultaneous two-electron transfer (ET) takes place. At early stages of O(2) binding the energy difference between the triplet and the singlet state is reduced by charge transfer (CT), which delocalizes the unpaired electrons and thus lowers the exchange stabilization onto the separated copper centers. The electron spins on the copper(II) ions are initially ferromagnetically coupled due to close to orthogonal magnetic orbital pathways through the dioxygen bridging ligand, and a change in the structure of the Cu(2)O(2) core turns on the superexchange coupling between the coppers. This favors the singlet state over the triplet state enabling intersystem crossing. Comparison with mononuclear model complexes indicates that the protein matrix holds the two copper(I) centers in close proximity, which enthalpically and entropically favors O(2) binding due to destabilization of the reduced binuclear site. This also allows regulation of the enthalpy by the change of the Cu--Cu distance in deoxyHc, which provides an explanation for the O(2) binding cooperativity in Hc. These results are compared to our earlier studies of Hemerythrin (Hr) and a common theme emerges where the spin forbiddeness of O(2) binding is overcome through delocalization of unpaired electrons onto the metal centers and the superexchange coupling of the metal centers via a ligand bridge.

A theoretical study on the origin of cooperativity in the formation of 3(10)- and alpha-helices.

Abstract: By using a simple repeating unit method, we have conducted a theoretical study which delineates the preferences for β-strand, 27-ribbon, 310-helix, and α-helix formation for a series of polyglycine models up to 14 amino acid residues (Ac-(Gly)n, n = 0, 1, 2, ..., 14). Interactions among residues, which result in cooperativity, are clearly indicated by variations in calculated energies of the residues. Whereas no cooperativity is found in the formation of β-strands and 27-ribbons, there is a significant cooperativity in the formation of 310- and α-helices, especially for the latter. In the case of α-helices, the 14th residue is more stable than the 3rd by about 3 kcal/mol. A good correlation between calculated residue energy and residue dipole moment was uncovered, indicating the importance of long-range electrostatic interactions to the cooperativity. The results of our calculations are compared with those of the AMBER and PM3 methods, and indicate that both methods, AMBER and PM3, need further developmen...

Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity.

Abstract: Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.

An enthalpic component in cooperativity: the relationship between enthalpy, entropy, and noncovalent structure in weak associations.

Abstract: Attempts to quantify binding interactions of noncovalent complexes in aqueous solution have been stymied by complications arising from enthalpy−entropy compensation and cooperativity. We have extended work detailing the relationship between noncovalent structure and free energy of binding to include the roles of enthalpy and entropy of association. On the basis of van't Hoff measurements of the dimerization of vancomycin type antibiotics, we demonstrate that positive cooperativity manifests itself in a more favorable enthalpy of association and a partially compensating less favorable entropy of association. Finally, we extend these results to rationalize thermodynamic observations in unrelated systems.

Propagating Conformational Changes Over Long (And Short) Distances in Proteins

Abstract: The problem of the propagation of conformational changes over long distances or through a closely packed protein is shown to fit a model of a ligand-induced conformational change between two protein states selected by evolution. Moreover, the kinetics of the pathway between these states is also selected so that the energy of ligand binding and the speed of the transition between conformational states are physiologically appropriate. The crystallographic data of a wild-type aspartate receptor that has negative cooperativity and a mutant that has no cooperativity but has native transmembrane signaling are shown to support this model.

Strong cooperativeness in the mononuclear iron(II) derivative exhibiting an abrupt spin transition above 400 K.

Abstract: The spin crossover system, [Fe(bzimpy)(2)](ClO(4))(2).0.25H(2)O, was reinvestigated above room temperature (bzimpy = 2,6-bis(benzimidazol-2-yl)pyridine). The system exhibits an abrupt low-spin to high-spin transition at T(c) = 403 K. Liberation of a fractional amount of water does not affect the spin crossover: the system is perfectly reversible with a hysteresis width of DeltaT = 12 K. The existence of the hysteresis at such high temperature determines that the lowest limit of the solid-state cooperativity parameter is J/k > 403 K despite long iron(II) separations (10 A). The high cooperativeness has been assigned to a perfect pi-stacking of the benzimidazole rings in the crystal lattice at a distance as short as 3.6 A. Variable-temperature IR data and the heat capacity measurements match well the magnetic data. The thermodynamic properties are DeltaH = 17 kJ mol(-)(1), DeltaS = 43 J K(-)(1) mol(-)(1), so that the entropy of the spin transition shows a considerable contribution from the molecular vibrations. A theoretical model has been applied in fitting the magnetic data along the whole hysteresis path. A statistical distribution of the cooperativity parameter led to the feature that angled walls of the hysteresis loop are well reproduced.

Long-range reactive dynamics in myoglobin.

Abstract: We report the complete vibrational spectrum of the probe nucleus 57Fe at the oxygen-binding site of the protein myoglobin. The Fe-pyrrole nitrogen stretching modes of the heme group, identified here, probe asymmetric interactions with the protein environment. Collective oscillations of the polypeptide, rather than localized heme vibrations, dominate the low frequency region. We conclude that the heme "doming" mode is significantly delocalized, so that distant sites respond to oxygen binding on vibrational time scales. This has ramifications for understanding long-range interactions in biomolecules, such as those that mediate cooperativity in allosteric proteins.

Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity.

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

In vitro stimulation of warfarin metabolism by quinidine: increases in the formation of 4'- and 10-hydroxywarfarin.

Abstract: It has been demonstrated that the activity of cytochrome P450 (CYP)3A4 in certain cases is stimulated by quinidine (positive heterotropic cooperativity). We report herein that the 4*- and 10hydroxylation of S- and R-warfarin are enhanced in human liver microsomal incubations containing quinidine. These reactions were catalyzed by CYP3A4, based on data derived from immunoinhibitory studies, with 4*-hydroxylation being preferentially associated with S-warfarin and 10-hydroxylation with R-warfarin. The 4*-hydroxylation of S-warfarin and 10-hydroxylation of R-warfarin increased with increasing quinidine concentrations and maximized at ;3- and 5-fold the values of controls, respectively. Stimulatory effects of quinidine also were observed with recombinant CYP3A4, suggesting that increases in warfarin metabolism were due to quinidine-mediated enhancement of CYP3A4 activity. This positive cooperativity of CYP3A4 was characterized by a 2.5-fold increase in Vmax for the 4*-hydroxylation of S-warfarin and a 5-fold increase in Vmax for the 10-hydroxylation of R-warfarin, with little change in Km values. Conversely, Vmax for the 3-hydroxylation of quinidine was not influenced by the presence of warfarin. These results are consistent with previous findings suggesting the existence of more than one binding site in CYP3A4 through which interactions may occur between substrate and effector at the active site of the enzyme. Such interactions were subsequently illustrated by a kinetic model containing two binding domains, and a good regression fit was obtained for the experimental data. Finally, stimulation of warfarin metabolism by quinidine was investigated in suspensions of human hepatocytes, and increases in the formation of 4*and 10-hydroxywarfarin again were observed in the presence of quinidine, indicating that this type of drug-drug interaction occurs in intact cells.

Cooperativity of binding epitopes and receptor chains in the BMP/TGFbeta superfamily.

Abstract: Bone morphogenetic proteins (BMP) are dimeric factors initiating several distinct signaling cascades by binding to two types of transmembrane serine/threonine kinase receptors (BRI and BRII), and are thus regulating several steps in embryonal development and adult tissue homeostasis. BMP-2 contains two symmetrical pairs of juxtaposed epitopes: the wrist epitope with high affinity to BRI consists of residues from both BMP-2 monomers, while the knuckle epitope resembles the low affinity site for BRII and comprises residues from only one monomer. Here we generated heterodimeric BMP-2 muteins with one monomer mutant in either epitope I for BRI (eI-) or epitope II for BRII (eII-) and the second monomer wild type for receptor interactions (m-). These muteins (B2eI-/B2m- and B2eII-/B2m-) were analyzed by biosensor analysis as well as by measuring their biological activity and compared to their homodimeric forms (either wild type or mutant). Depletion of only one epitope II results in the loss of biological activity as measured byalkaline phosphatase (ALP) activity and Smad induced reportergene assays. However, depletion of only one epitope I shows a reduction of ALP activity to about 25%, while the activation of the Smad pathway remained normal. Homomeric muteins are non-functional for both Smad and ALP activation. This suggests that two functional epitopes II have to be present on one BMP-2 molecule for receptor activation. Futhermore, both pathways (Smad and ALP) are triggered differently by distinct BMP-receptor complexes. Heteromeric BMP-2 mutants therefore allow a distinguishable manipulation of either pathway and thus represent important tools for the generation of specific BMP-2 antagonists or agonists.