Substitution at residue 214 of human thymidylate synthase alters nucleotide binding and isomerization of ligand-protein complexes

Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089-7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme-substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, Kiso, the ratio of the forward rate of isomerization (kiso) to the reverse rate of isomerization (kr, iso), is 2-6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on kiso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.     

Flow cytometric competitive binding assay for determination of actinomycin-D concentrations

A single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin-D for common binding sites on DNA coated 10 microns diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin-D concentration.     

A universal homogeneous assay for high-throughput determination of binding kinetics

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug–target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR–FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein–protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

Solid-phase preparation of protein complexes

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.     

The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments

A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.     

Accelerated dissociation of estrogen receptor--ligand complexes by estradiol. Evidence for negative cooperativity of binding

The rate of dissociation of 17β-[³H]estradiol that had been previously equilibrated to a low degree of saturation of immature rat uterine cytoplasmic estrogen receptor was shown to increase over 40-fold in the presence of additional ligand. This effect was specific for either labeled or unlabeled estradiol, was observed under conditions in which the rebinding of dissociated ligand was shown not to occur, was distinguishable from the activation of cytoplasmic receptor, and was dependent upon the degree of saturation of the receptor by ligand. It occurred under conditions in which the receptor population was apparently uniform and stable and utilized an assay method that is particularly sensitive to low concentrations of cytosol protein. Once saturation of the receptor attained 15% of the available ligand binding sites, further increases of the dissociation rate of receptor-ligand complex could not be produced by the inclusion of additional estradiol. It was shown that exchange of ligand molecules in given binding sites was unlikely. Rather, support was given to the hypothesis that interactions were occurring between separate binding sites in the receptor population. The decrease of the apparent affinity of receptor for ligand when the fractional saturation of receptor increases has been defined as negative cooperativity. It is proposed that this phenomenon may be significant in the regulation of the response of target cells to estrogens.     

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (K_d) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.     

The rates of formation and dissociation of actin-myosin complexes. Effects of solvent, temperature, nucleotide binding and head-head interactions.

The rates of formation and dissociation of actin-subfragment 1 and actin-heavy mero-myosin complexes were measured by using light-scatter and the change in fluorescence of N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine (IAEDANS)-labelled acting as probes. Association rate measurements were made at low protein concentration, where the transients approximated to single exponentials with rate constants proportional to the concentration of reactant in excess. Dissociation rate measurements were made by displacing IAEDANS-actin from myosin with excess native actin and by a salt jump. The second-order rate constant of association for actin-subfragment 1 was 3 x 10(6) M-1 . s-1 in 60 mM-KCl at 13 degree C. It was decreased 10-fold in 500 mM-KCl and in 50% (v/v) glycol. It was decreased 6-fold when MgADP or Mg[beta gamma-imido]ATP bound to myosin. The dissociation rate constant was 0.012 s-1 in 60 mM-KCl at 13 degree C. It was increased 4-fold by 500 mM-KCl, 25-fold by 50% glycol, 8-fold by MgADP binding and 170-fold by Mg[beta gamma-imido]ATP binding. Ea for association was 70 kJ . mol-1 and for dissociation 35 kJ . mol-1. Heavy meromyosin associated at twice the rate observed for subfragment 1 and dissociated at less than one-twentieth of the rate for subfragment 1 (60 mM-KCl, 25 degree C), but when Mg[beta gamma-imido]ATP bound actin-heavy meromyosin dissociated at one-half the rate for subfragment 1. There were significant correlations between increase in the dissociation rate constant, decrease in binding constant and increase in magnitude of conformational change. The association rate constant did not correlate with any property of the actin-myosin complex.     

The architecture of the binding site in redox protein complexes: implications for fast dissociation

Interprotein electron transfer is characterized by protein interactions on the millisecond time scale. Such transient encounters are ensured by extremely high rates of complex dissociation. Computational analysis of the available crystal structures of redox protein complexes reveals features of the binding site that favor fast dissociation. In particular, the complex interface is shown to have low geometric complementarity and poor packing. These features are consistent with the necessity for fast dissociation since the absence of close packing facilitates solvation of the interface and disruption of the complex.     

On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to “fold” to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.     

Equilibrium binding constants and facile dissociation of novel serum albumin-dicyanoaurate(I) complexes

 Dicyanoaurate(I), Au(CN)₂ ^–, an important metabolite of chrysotherapy agents (anti-arthritic gold drugs), contains two tightly bound cyanide ligands which render it relatively unreactive toward ligand exchange reactions with potential gold-binding ligands. The extent and nature of its binding to bovine serum albumin (BSA), which may modulare the in vivo activity of Au(CN)₂ ^–, were investigated to determine whether Au(CN)₂ ^– might be more bioavailable than other gold complexes. ¹³C NMR spectroscopy, radioisotope tracers, chromatography, ultrafiltration, and atomic spectroscopy, employing Au(¹³CN)₂ ^– or Au(¹⁴CN)₂ ^– as appropriate, revealed two distinct binding mechanisms. The dominant reaction is reversible association (non-specific binding) of intact Au(CN)₂ ^– ions to form BSA·[Au(CN)₂ ^–] _n adducts. Approximately one equivalent binds with an equilibrium binding constant (pH 7.4, 25  °C) of K ₁=5.5 (±1.1)×10⁴, and three additional equivalents bind with a constant of 7.0 (±0.1)×10³. Au(¹³CN)₂ ^– associated with albumin is characterized by a broad ¹³C NMR resonance at δ_C=154.7 ppm compared to the sharp resonance of the free complex at 156.4 ppm. The BSA·[Au(CN)₂ ^–] _n adducts readily dissociate during gel exclusion chromatography and are therefore underestimated, but are retained and accurately quantitated by ultrafiltration methods. The second binding mechanism is a ligand exchange reaction at Cys-34, to form AlbSAuCN, which accounts for only a small fraction (≤11%) of the bound gold. The small extent of the latter interaction differentiates Au(CN)₂ ^– from the gold drugs such as auranofin, aurothiomalate (Myochrysin) and aurothioglucose (Solganol), which undergo ligand exchange at Cys-34 of albumin to form tightly bound gold-protein complexes. The weak interaction at Cys-34 and the facile dissociation of bound, intact Au(CN)₂ ^– are consistent with its putative role as a gold metabolite that can be accumulated intracellularly. Received: 2 July 1997 / Accepted: 24 September 1997     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to K_V2.1 channels is inhibited by another K_V2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel Na_V1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-K_V2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl K_V channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of K_V channels can be selectively targeted by small molecules to modify channel function.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.