Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.     

Depression of Neuronal Protein Synthesis Initiation by Protein Tyrosine Kinase Inhibitors

Abstract— Growth factors stimulate cellular protein synthesis, but the intracellular signaling mechanisms that regulate initiation of mRNA translation in neurons have not been clarified. A rate-limiting step in the initiation of protein synthesis is the formation of the ternary complex among GTP, eukaryotic initiation factor 2 (elF-2), and the initiator tRNA. Here we report that genistein, a specific tyrosine kinase inhibitor, decreases tyrosine kinase activity and the content of phosphotyrosine proteins in cultured primary cortical neurons. Genistein inhibits protein synthesis by >80% in a dose-dependent manner (10–80 μg/ml) and concurrently decreases ternary complex formation by 60%. At the doses investigated, genistein depresses tyrosine kinase activity and concomitantly stimulates PKC activity. We propose that a protein tyrosine kinase participates in the initiation of protein synthesis in neurons, by affecting the activity of elF-2 directly or through a protein kinase cascade.     

The hydroxylation of phenylalanine and tyrosine: a comparison with salicylate and tryptophan.

The hydroxylation of phenylalanine by the Fenton reaction and gamma-radiolysis yields 2-hydroxy-, 3-hydroxy-, and 4-hydroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2,3- and 3,4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and the presence or absence of oxidants. During gamma-radiolysis and the Fenton reaction the same hydroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hydroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimerization reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine show a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pH 8 is due to the rapid oxidation of dopa to melanin. The relative abilities of iron chelates (HLFe(II) and HLFe(III) to promote hydroxyl radical formation from hydrogen peroxide are nitrilotriacetate (nta) greater than ethylenediaminediacetate (edda) much greater than hydroxyethylethylenediaminetriacetate greater than citrate greater than ethylenediaminetetraacetate greater than diethylenetriaminepentaacetate greater than adenosine 5'-triphosphate greater than pyrophosphate greater than adenosine 5'-diphosphate greater than adenosine 5'-monophosphate. The high activity of iron-nta and -edda chelates is explained by postulating the formation of a ternary Fe(III)-L-dopa complex in which dopa reduces Fe(III). The hydroxylations of phenylalanine and tyrosine are similar to that of salicylate (Z. Maskos, J. D. Rush, and W. H. Koppenol, 1990, Free Radical Biol. Med. 8, 153-162) and tryptophan (preceding paper) in that oxidants augment the formation of hydroxylated products by catalyzing the dismutation of hydroxyl radical adducts to the parent compound and a stable hydroxylated product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production.     

Solution interactions between the uranyl cation [UO2(2+)] and histidine, N-acetyl-histidine, tyrosine, and N-acetyl-tyrosine

Complexes of the uranyl cation [UO(2)(2+)] with histidine (His), N-acetyl-histidine (NAH), tyrosine (Tyr), and N-acetyl-tyrosine (NAT) were studied by UV-visible and NMR spectroscopy, and by potentiometric titration. Protonation constants for each ligand are reported, as are cumulative formation constants for uranyl-amino acid complexes. Coupling constant data (J(CH)) for uranyl-histidine complexes indicate that inner-sphere solution interactions between histidine and uranyl cation are solely at the carboxylate site. At 25 degrees C the major uranyl-histidine complex has a cumulative formation constant of logbeta(110)=8.53, and a proposed formula of [UO(2)HisH(2)(OH)(2)](+); the stepwise formation constant, logK(UL), is estimated to be 5.6 ( approximately 8.53-(-6.1)-(-6.1)-15.15). Outer-sphere interactions, H-bonding or electrostatic interactions, are proposed as contributing a significant portion of the stability to the ternary uranyl-hydroxo-amino acid complexes. The temperature dependent protonation constants of histidine and formation constants between uranyl cation and histidine are reported from 10 to 35 degrees C; at 25 degrees C, DeltaG=-43.3 kJ/mol.     

Heparin promotes the binding of thrombin to fibrin polymer. Quantitative characterization of a thrombin-fibrin polymer-heparin ternary complex

The binding of human alpha-thrombin (IIa) to fibrin polymer (FnIIp) was studied in the presence and absence of a high affinity 20,300 Mr heparin (H) at pH 7.4, I 0.15, and 23 degrees C. In the absence of heparin, thrombin interacts with a high affinity class of binding sites on fibrin polymer with a dissociation constant of 301 +/- 36 nM in a manner which is independent of the enzyme active site. Studies of thrombin binding as a function of heparin and fibrin polymer concentrations imply that a ternary thrombin-fibrin polymer-heparin complex (IIa.FnIIp.H) is formed. Assembly of the ternary complex occurs randomly through the interactions of all three possible intermediate binary complexes; IIa.H, IIa.FnIIp, and FnIIp.H. Using an independently determined value of 280 +/- 35 nM for the FnIIp.H dissociation constant, global fits of the binding data yield a dissociation constant of 15 +/- 6 nM for the IIa.H interaction and 47 +/- 9 nM for the IIa.H intermediate binary complex interaction with FnIIp. These studies indicate that heparin enhances the binding of thrombin to fibrin polymer 6.4-fold with an overall dissociation constant for ternary complex formation of 705 nM2. The effect of heparin molecular weight on ternary complex formation has also been investigated. Heparins of molecular weights 11,200-20,300 behave similarly with respect to their influence on ternary complex formation, whereas heparins of lower molecular weight are less effective in promoting thrombin binding to fibrin polymer. This effect of heparin is also independent of whether it has high or low affinity for antithrombin III. The demonstration of the formation of a ternary IIa.FnIIp.H complex complements kinetic evidence indicating the formation of an analogous ternary complex with fibrin II monomer (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623). The possible implications of these findings for the in vivo distribution and actions of thrombin and the clinical efficacy of heparin are also discussed.     

Direct Evidence of an Elongation Factor-Tu/Ts·GTP·Aminoacyl-tRNA Quaternary Complex

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA^Phe ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917–13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA^aa. Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.     

Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells

A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway.     

Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate-aminoacyl-tRNA complexes

A new method for measuring the dissociation rate of the Escherichia coli elongation factor Tu-GTP--aminoacyl-tRNA complex has been developed and applied to the determination of the dissociation rates of ternary complexes formed between E. coli EF-Tu-GTP and a set of E. coli aminoacyl-tRNAs. The set of aminoacyl-tRNAs includes at least one tRNA coding for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. The results reveal that the dissociation rates vary for each ternary complex. Tu-GTP-Gln-tRNA dissociates the slowest and Tu-GTP-Val-tRNA the fastest of all noninitiator ternary complexes at 4 degrees C, pH 7.4. The equilibrium dissociation constant for Tu-GTP-Thr-tRNA has been determined to be 1.3 (0.4) X 10(-9) M under identical reaction conditions, and the absolute value of the equilibrium dissociation constant has been calculated for 28 ternary complexes from the relative equilibrium dissociation constant ratios previously measured [Louie, A., Ribeiro, N. S., Reid, B. R., & Jurnak, F. (1984) J. Biol. Chem. 259, 5010-5016]. The association rate of each ternary complex has been estimated from the ratio of the dissociation rate relative to the equilibrium dissociation constant. Tu-GTP-His-tRNA associates the fastest and Tu-GTP-Leu-tRNA1Leu the slowest. By inclusion of Tu-GTP-Met-tRNAfMet in the studies, evidence has been obtained that suggests that the initiator ternary complex does not function in the elongation cycle because the dissociation rate of the complex is very fast.     

Tryptophan-free human PNP reveals catalytic site interactions

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid–base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR–Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions

The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.     

Design,synthesis, and evaluation of novel N-1 fluoroquinolone derivatives: Probing for binding contact with the active site tyrosine of gyrase

Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.     

Properties of a defined mutant of Escherichia coli thymidylate synthase

A mutant of Escherichia coli thymidylate synthase (F3-TS), resulting from the replacement of a tyrosine for a cysteine 50 amino acids from the amino-terminal end, has been purified to homogeneity and found to contain less than 0.2% of the activity of the native enzyme (thyA-TS). Although this protein formed a ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate, like the native enzyme, the extent of complex formation was significantly impaired as determined by equilibrium dialysis and circular dichroism. Thus, unlike the native enzyme, where 2 mol of FdUMP were present in each mole of ternary complex, F3-TS contained less than 1 mol of FdUMP/mol of ternary complex. Similarly, the binding of dUMP by F3-TS was greatly diminished relative to thyA-TS, but its binding as well as that of FdUMP could be improved by the presence of either the folate substrate or a tight binding folate analogue, 10-propargyl-5,8-dideazafolate (PDDF). However, despite the fact that PDDF enhanced the binding of FdUMP and dUMP to F3-TS, the binding of PDDF to the mutant enzyme was also greatly impaired. This contrasts with the native enzyme, which, under the same conditions, bound about 2 mol of PDDF/mol of enzyme in the presence or absence of either FdUMP or dUMP. Circular dichroism analyses with PDDF in the presence of dUMP or FdUMP yielded analogous results, but the effects were less dramatic than those obtained by equilibrium dialysis. Evidence in support of a structural difference between thyA-TS and F3-TS was obtained by demonstrating that the latter protein was 15-fold slower in forming a ternary complex with dUMP and PDDF than the former and that the mutant enzyme was less stable than the native enzyme.     

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

DNA binding induces conformational transition within human DNA topoisomerase I in solution

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.     

A dimeric ternary complex of FGFR [correction of FGFR1], heparin and FGF-1 leads to an 'electrostatic sandwich' model for heparin binding.

BACKGROUND: Fibroblastic growth factors (FGFs) are a family of cytokines involved in regulation of cell growth, differentiation and chemotaxis in a variety of tissue types. High-affinity FGF receptors (FGFRs) are transmembrane proteins that consist of three extracellular immunoglobulin-like domains, a transmembrane helix and an intracellular protein tyrosine kinase signalling domain. FGFRs are activated through ligand-dependent dimerization that allows trans-autophosphorylation of the tyrosine kinase domains. Heparin or heparin-like molecules, such as heparan sulphate proteoglycans, bind to both FGFs and FGFRs and are required for FGF signal transduction. At present no structure of the ternary complex for FGFR, FGF and heparin exists. RESULTS: We have used the type-1 interleukin-1 receptor-interleukin-1 beta complex crystal structure, in which both the ligand and the receptor are homologous to those of the FGF-FGFR pair, to identify potential interactions in the FGFR-heparin-FGF ternary complex. A key feature of the modelled complex is the 'electrostatic sandwich' that is formed between the positively charged surfaces of FGF and the receptor, with the negatively charged heparin captured in between. The ternary complex places limits on the range of likely modes of receptor dimerization: one of five different dimeric receptor complexes built from the ternary complex correlates best with the experimental data. CONCLUSIONS: The ternary complex of FGFR, FGF and heparin, derived on the basis of the homologous interleukin-1 receptor complex, is in agreement with much of the published experimental data, as is the dimeric receptor complex (FGFR-heparin-FGF)2. This work suggests that the FGF interactions seen in crystal structures, which have previously been used to predict the mode of FGF dimerization, might not be relevant to the biologically active dimeric FGFR-heparin-FGF complex.     

Transmission of ADP.vanadate-induced conformational changes to three peptide segments of myosin subfragment-1

In order to study the conformational changes associated with formation of the stable ternary complex of myosin subfragment-1 (S-1) with ADP and orthovanadate (Vi), S-1 was fluorescently labeled with 9-anthroylnitrile, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and 5-(iodoacetamido) fluorescein at the 23-, 50-, and 20-kDa peptide segments of S-1, respectively (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194; Hiratsuka, T. (1986) J. Biol. Chem. 261, 7294-7299; Takashi, R. (1979) Biochemistry 18, 5164-5169). The extrinsic fluorescence of these S-1 derivatives was sensitive not only to binding of ADP but to formation of the stable ternary complex with ADP and Vi. By using these fluorescent properties, the kinetics of formation of the stable ternary complexes of these S-1 derivatives with ADP and Vi, M. ADP.Vi, were analyzed according to the scheme proposed by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). [Formula; see text] The values obtained for KVi )0.2-0.4 mM) and k (0.03-0.05 s-1) of these S-1 derivatives were similar regardless of the peptide segments of S-1 where the fluorophore had been covalently labeled. These results suggest that the conformational changes, which are induced by formation of the stable ternary complex of S-1 with ADP and Vi, are transmitted to all three peptide segments of S-1 at a similar rate. The present results also encourage us to confirm that the ATPase site of S-1 resides at or near the region where all three peptide segments of S-1 are contiguous.     

Glycosaminoglycans inhibit formation of the 140 kDa insulin-like growth factor-binding protein complex.

The 140 kDa insulin-like growth factor (IGF)-binding protein complex in human serum consists of three subunits: an acid-labile, non-IGF-binding glycoprotein (alpha-subunit), an IGF-binding glycoprotein known as BP-53 or IGFBP-3 (beta-subunit), and IGF-I or IGF-II (gamma-subunit). This study investigates the regulation, by salt and glycosaminoglycans, of ternary (alpha-beta-gamma) complex formation, measured by incubating radioiodinated alpha-subunit with a mixture of IGF-I and IGFBP-3 and precipitating bound radioactivity with an anti-IGFBP-3 antiserum. Increasing NaCl concentrations progressively decreased ternary complex formation without any effect on binary (beta-gamma) complex formation. In 0.15 M-NaCl, the association constant for the ternary complex was 0.318 +/- 0.092 nM-1, 100-fold lower than that for the binary complex. Glycosaminoglycans also inhibited ternary complex formation without affecting the binary complex. Heparin [50% inhibition at 0.27 +/- 0.08 units/ml (1.5 +/- 0.4 micrograms/ml)] was more potent than heparan sulphate (50% inhibition at 15 +/- 7 micrograms/ml), with chondroitin sulphate even less potent. The inhibition by heparin was due principally to a decrease in binding affinity, from 0.604 +/- 0.125 to 0.151 +/- 0.024 nM-1 in the presence of 0.25 units of heparin/ml, with a slight decrease in the number of apparent binding sites from 1.05 +/- 0.08 to 0.85 +/- 0.15 mol of alpha-subunit bound/mol of beta-subunit. Since the ternary IGF-binding protein complex cannot cross the capillary barrier, it is proposed that a decrease in the affinity of the complex, mediated by circulating or cell-associated glycosaminoglycans, may be important in the passage of IGFs and IGFBP-3 to the tissues.