Phosphate ion partially relieves the cooperativity of effector binding in D-3-phosphoglycerate dehydrogenase without altering the cooperativity of inhibition

The binding of L-serine to phosphoglycerate dehydrogenase from E. coli displays elements of both positive and negative cooperativity. In addition, the inhibition of enzymatic activity by L-serine is also cooperative with Hill coefficients greater than 1. However, phosphate buffer significantly reduces the cooperative effects in serine binding without affecting the cooperativity of inhibition of activity. The maximal degree of inhibition and fluorescence quenching in Tris buffer occurs when an average of two serine binding sites out of four are occupied. This value increases to three out of the four sites at maximal levels of inhibition and quenching in phosphate buffer. The increase from two to three sites appears to be due to the ability of phosphate to reduce the site to site cooperative effects and render each ligand binding site less dependent on each other. The correlation between the level of inhibition and the fractional site occupancy indicates that in Tris buffer, one serine is bound to each interface at maximal effect. In the presence of phosphate, the order of binding appears to change so that both sites at one interface fill before the first site at the opposite interface is occupied. In each case, there is a good correlation between serine binding, conformational change at the regulatory site interfaces, and inhibition of enzyme activity. The observation that phosphate does not appear to have a similar effect on the cooperativity of inhibition of enzymatic activity suggests that there are two distinct cooperative pathways at work: one path between the four serine binding sites, and one path between the serine binding sites and the active sites.     

Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding

The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.     

Hybrid tetramers reveal elements of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when l-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by approximately 58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.     

Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers

A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.     

Hydrophobic agaroses: basis for a model of multivalent effector-receptor interactions.

An increase in the density of butyl residues bound to Sepharose 4B leads to an enhancement of the affinity of these gels for phosphorylase b in the presence of 1.1M ammonium sulfate. A Hill coefficient of 2.9 indicates that a minimum of ca. 3 binding sites is involved in the positive cooperative adsorption of this enzyme. Binding studies of phosphorylase b on butyl-Sepharose of a specific degree of substitution demonstrate that the affinity of the gel for this ligand decreases as a function of fractional saturation. A Hill coefficient of 0.44 indicates negative cooperativity as a result of multivalent binding. From these observations a multivalent, mobile receptor model is derived which can explain such characteristics of effector-receptor interactions as: positive and negative cooperativity, high binding constants and low dissociation rate constants. The application of this model to experiments taken from the literature on the binding of the multivalent effectors concanavalin A and cholera toxin to fat cells shows that the postulated mode of interactions is probably realized in nature.     

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.     

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

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.     

How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.     

Binding properties of ferrocene-glutathione conjugates as inhibitors and sensors for glutathione S-transferases

The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.     

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.     

Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis

Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.     

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).     

Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding

Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes has been investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite seven intrachain disulfide links, it is shown that some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association has been evaluated. Data are consistent with a model in which the first mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the second mole of ligand induces most of the described conformational change. Accordingly, the positive cooperativity between the two dimeric binding sites is linked mainly to the binding of the second ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.     

Evidence for the reversible binding of paraquat to deoxyribonucleic acid

Evidence for the reversible binding of paraquat to calf thymus DNA has been obtained using equilibrium dialysis and thermal melting point determinations. The data indicated the presence of at least two populations of binding site with affinity constants of 6.2 X 10(4) and 7.1 X 10(3) M-1, respectively. The binding capacities of DNA for paraquat were 66 and 480 nmol/mumol DNA nucleotide, respectively, and were equivalent to one ligand bound per 2 DNA phosphate groups. Putrescine inhibited paraquat binding to the low affinity sites without altering binding to the high affinity sites. Scatchard plots of paraquat binding characteristics indicated the presence of positive cooperativity between the compound and DNA. Thermal melting curves of DNA in the presence of paraquat and the endogenous amines putrescine, spermidine and spermine, provided evidence that paraquat cross-linked to DNA with a similar affinity as spermidine. The thermal melting point data also suggested the presence of positive cooperativity between ligand and macromolecule that possibly resulted from a conformation change in the structure of the DNA molecule. Paraquat competitively inhibited the binding of ethidium bromide to DNA and this effect was reversed by Na+. From the data, it is suggested that paraquat binds primarily to the negatively charged phosphates on the DNA backbone but is displaced into the interbase region occupied by the intercalator ethidium bromide. DNA binding of paraquat may, in part, account for its weak mutagenic activity.     

Thermodynamic model of cooperativity in a dimeric protein: unique and independent parameters formulation.

A model of the cooperative interaction of ligand binding to a dimeric protein is presented based upon the unique and independent parameters (UIP) thermodynamic formulation (Gutheil and McKenna, Biophys. Chem. 45 (1992) 171-179). The analysis is developed from an initial model which includes coupled conformational and ligand binding equilibria. This completely general model is then restricted to focus on conformationally mediated cooperative interactions between the ligands and the expressions for the apparent ligand binding constant and the apparent ligand-ligand interaction constant are derived. The conditions under which there is no cooperative interaction between the ligands are found as roots to a polynomial equation. Consideration of the distribution of species among the various conformational states in this general model leads to a set of inequalities which can be represented as a two dimensional plot of boundaries. By superimposing a contour plot of the value of the apparent ligand-ligand interaction constant over the plot of boundaries a complete graphical representation of this system is achieved similar to a phase diagram. It is found that the parameter space homologous to Koshland-Nemethy-Filmer type of model is most consistent with both positive and negative cooperativity in this model. The maximal amount of positive and negative cooperativity are found to be simple functions of Kc, the equilibrium constant associated with the change of a subunit and ligand from the unligated to ligated conformation. It is shown that under certain limiting conditions the apparent allosteric interaction between ligands is equal to the conformational interaction between subunits. The methods presented are generally applicable to the theoretical analysis of thermodynamic interactions in complex systems.     

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase

F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.     

Cooperativity and noncooperativity in the binding of NAD analogues to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Using NAD analogues as ligands, the structural requirements for negative cooperativity in binding to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase were examined. Although the affinity of nicotinamide hypoxanthine dinucleotide is considerably lower than that of NAD+, it also binds to the enzyme with negative cooperatively. Two pairs of nicotinamide hypoxanthine dinucleotide binding sitess were distinguished, one pair having an affinity for the analogue which is 15 times that of the second pair. Negative cooperativity is also found in the Km values for the analogue. Thus modification of the adenine ring of NAD+ to hypoxanthine does not abolish negative cooperativity in coenzyme binding. Adenosine diphosphoribose binding to the same enzyme shows neither positive nor negative cooperativity, indicating that cooperativity apparently requires an intact nicotinamide ring in the coenzyme structure, under the conditions of these experiments. Occupancy of the nicotinamide subsite of the coenzyme binding site is not necessary for half-of-sites reactivity of alkylating or acylating compounds (Levitzki, A. (1974), J. Mol, Biol. 90, 451-458). However, it can be important in the negative cooperativity in ligand binding, as illustrated by adenosine diphosphoribose which fails to exhibit negative cooperativity. Occupancy of the adenine subsite by adenine is important for stabilization of the enzyme against thermal denaturation. Whether the stabilization is due to an altered conformation of the subunits or stabilization of the preexisting structure of the apoenzyme cannot be determined from these studies. However, nicotinamide hypoxanthine dinucleotide does not contribute to enzyme stability although it serves as a substrate and shows negative cooperativity.     

Insulin receptor binding kinetics: modeling and simulation studies

Biological actions of insulin regulate glucose metabolism and other essential physiological functions. Binding of insulin to its cell surface receptor initiates signal transduction pathways that mediate cellular responses. Thus, it is of great interest to understand the mechanisms underlying insulin receptor binding kinetics. Interestingly, negative cooperative interactions are observed at high insulin concentrations while positive cooperativity may be present at low insulin concentrations. Clearly, insulin receptor binding kinetics cannot be simply explained by a classical bimolecular reaction. Mature insulin receptors have a dimeric structure capable of binding two molecules of insulin. The binding affinity of the receptor for the second insulin molecule is significantly lower than for the first bound insulin molecule. In addition, insulin receptor aggregation occurs in response to ligand binding and aggregation may also influence binding kinetics. In this study, we develop a mathematical model for insulin receptor binding kinetics that explicitly represents the divalent nature of the insulin receptor and incorporates receptor aggregation into the kinetic model. Model parameters are based upon published data where available. Computer simulations with our model are capable of reproducing both negative and positive cooperativity at the appropriate insulin concentrations. This model may be a useful tool for helping to understand the mechanisms underlying insulin receptor binding and the coupling of receptor binding to downstream signaling events.     

Cooperative interaction between developmentally regulated troponin T and tropomyosin isoforms in the absence of F-actin

Troponin T (TnT) is the tropomyosin (Tm) binding subunit of the troponin complex that mediates the Ca(2+) regulation of actomyosin interaction in striated muscles. Troponin T isoform diversity is marked by a developmentally regulated acidic to basic switch that may modulate muscle contractility. We previously reported that transgenic expression of fast skeletal muscle TnT altered the cooperativity of cardiac muscle. In the present study, we have demonstrated that the binding of acidic TnT to troponin I is weaker than that of basic TnT. However, affinity chromatography experiments showed that Tm bound to acidic TnT with a greater affinity than to basic TnT, consistent with the significantly higher maximal binding of acidic TnT to Tm in solid phase binding assays. Competition and co-immunoprecipitation experiments demonstrated that the binding of TnT to Tm was cooperative in the absence of F-actin. The cooperativity between TnT molecules for Tm binding can be initiated by the conserved COOH-terminal T2 fragment of TnT. This indicates that the interaction of TnT with Tm induces a conformational change in Tm, promoting interaction of TnT with adjacent Tm dimers. This finding suggests a role for TnT and its acidic and basic isoforms in the cooperative release of the inhibition of striated muscle actomyosin interaction.     

Models for binding cooperativities of inhibitors with transthyretin

Here, molecular dynamics (MD) simulations are performed to study the differences of binding channel shapes of TTR with two inhibitors, flufenamic acid (FLU) and one kind of N-phenyl phenoxazine (BPD). The asymmetries of global structure including the central binding channel are found to be intrinsic. Moreover, the conformational changes of the binding channel are responsible for negative cooperativity (NC) or independent cooperativity (IC) of ligands. The results suggested a possible binding mechanism addressing NC of FLU and IC of BPD. For FLU, when the first ligand binds with TTR, it leads to expansion of the second binding site which may weaken the interaction of the second FLU with TTR. But for BPD, the first ligand's binding changes the second site's shape slightly, the second ligand has similar binding ability with TTR in the second site like the first binding event.