Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.     

Release of iron from transferrin by phosphonocarboxylate and diphosphonate chelating agents

The rates at which phosphonocarboxylate and diphosphonate ligands remove iron from the serum iron transport protein transferrin at 25 degrees C and pH 7.4 have been evaluated. These ligands show a combination of saturation and first-order kinetics with respect to the free ligand concentrations. The ability of the ligands to remove iron from transferrin appears to be subject to steric restrictions that are essentially identical to those associated with the ability of a ligand to substitute for the synergistic carbonate anion. This observation supports the hypothesis that the first-order component for iron removal involves a mechanism in which the rate-limiting step is the slow substitution of the synergistic carbonate by the incoming chelating agent. Studies on monoferric transferrins indicate that phosphonocarboxylates are unusually effective at removing iron from the C-terminal site of the protein. Difference UV spectroscopy has been used to show that the phosphonocarboxylates bind strongly to apotransferrin. It is suggested that the rapid release of iron from the C-terminal site may be due to the binding of the ligand to an allosteric anion-binding site in the C-terminal lobe of the protein.     

Surface plasmon resonance as a probe of protein isomerization

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.     

Autocrine ligand binding to cell receptors. Mathematical analysis of competition by solution "decoys".

Autocrine ligands have been demonstrated to regulate cell proliferation, cell adhesion, and cell migration in a number of different systems and are believed to be one of the underlying causes of malignant cell transformation. Binding of these ligands to their cellular receptors can be compromised by diffusive transport of ligand away from the secreting cell. Exogenous addition of antibodies or solution receptors capable of competing with cellular receptors for these autocrine ligands has been proposed as a means of inhibiting autocrine-stimulated cell behavioral responses. Such "decoys" complicate cellular binding by offering alternative binding targets, which may also be capable of aiding or abating transport of the ligand away from the cell surface. We present a mathematical model incorporating autocrine ligand production and the presence of competing cellular and solution receptors. We elucidate effects of key system parameters including ligand diffusion rate, binding rate constants, cell density, and secretion rate on the ability of solution receptors to inhibit cellular receptor binding. Both plated and suspension cell systems are considered. An approximate analytical expression relating the key parameters to the critical concentration of solution "decoys" required for inhibition is derived and compared to the numerical calculations. We find that in order to achieve essentially complete inhibition of surface receptor binding, the concentration of decoys may need to be as much as four to eight orders of magnitude greater than the equilibrium disociation constant for ligand binding to surface receptors.     

Failure of oligosaccharide MOPC-104E IgM complexes to bind C1q and to activate C1

The capacity of anti-dextran MOPC-104E IgM to bind and activate the first complement component (C1) in the presence of various specific monovalent oligosaccharides was investigated. Enzyme-linked immunosorbent assay revealed that IgM-oligosaccharide complexes saturated up to 97% with ligands were not capable of binding C1q under physiological conditions. Nor was any activation of reconstituted C1 observed. These results indicate that occupation of the single IgM binding sites by a monovalent ligand is not sufficient to induce a signal for complement activation.     

Mixed extracellular matrix ligands synergistically modulate integrin adhesion and signaling

Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor alpha(2)beta(1) and the fibronectin (FN) receptor alpha(5)beta(1) to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling.     

Studies on the structure and properties of nickel complexes in a set of amide-based 13-membered macrocyclic ligands

This work reports a systematic investigation to understand the structural, spectroscopic and redox properties of Ni(II) ion in a set of 13-membered amide-based macrocyclic ligands. Four macrocyclic ligands containing e⁻-donating/withdrawing substituents and their Ni(II) complexes have been synthesized and characterized. Structural analysis shows that the macrocyclic ligands create a square-planar environment and nicely accommodate the Ni(II) ion. Electrochemical results suggest that the complexes are capable of undergoing metal-centered oxidation. The electron-donating substituents on ligand lowers the redox potentials and better stabilizes the +3 oxidation state of metal. The electrochemically generated Ni^III species are shown to have rich spectroscopic features. For majority of complexes, the oxidized species are concluded to be Ni^III by their anisotropic EPR spectra typical for Ni^III ion in square-planar geometry. The absorption and EPR spectra for nickel complex bearing an -OMe group on the ligand; however, suggest a Ni(II) complex with a ligand-based radical.     

Molecular and cellular mechanisms of receptor-mediated endocytosis.

In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors and ligands are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of certain viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Although endocytosis is common to all nucleated eukaryotic cells, the factors that regulate these receptor-mediated endocytic pathways are not fully understood. Defective receptors that are not capable of undergoing normal endocytosis can lead to certain disease states, as in the case of familial hypercholesteremia (FH). This review has three objectives: (i) to describe the different routes that receptors and ligands follow after internaliation; (ii) to describe the potential mechanisms which regulate the initiation and subsequent sorting of receptors and ligands so they reach their final destination; and (iii) to describe the potential functions of receptor-mediated endocytosis.     

Analysis of binding modes of ligands to multiple conformations of CYP3A4

Cytochromes P450 (CYPs) are extremely versatile enzymes capable of catalyzing a vast number of compounds, and CYP3A4 is no exception metabolizing approximately half of the currently marketed drugs, besides endogenous compounds. To metabolize such a variety of compounds, CYP3A4 has to be extremely flexible, which makes interaction studies difficult. We employ a multi-conformational docking setup where conformations are generated by several molecular dynamics simulations to analyze the binding modes of various ligands, and the docking is considered successful if the ligand site of catalysis (SOC) is within 6.0 Å of the haem Fe. While docking with the X-ray structure proved unsuccessful with all ligands, the multi-conformational docking achieved successful binding of each ligand to at least one protein conformation. Analysis of the docked solutions highlights residues in the active site cavity that may have an important role in access, binding and stabilization of the ligand.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.     

Structural study of biologically significant ligands with major birch pollen allergen Betv1 by docking and molecular dynamics simulation

The major birch pollen allergen, Betv1 of Betula verrucosa is the main causative agent of birch pollen allergy in humans. Betv1 is capable of binding several physiological ligands including fatty acids, flavones, cytokinins and sterols. Until now, no structural information from crystallography or NMR is available regarding binding mode of any of these ligands into the binding pocket of Betv1. In the present study thirteen ligands have been successfully docked into the hydrophobic cavity of Betv1 and binding free energies of the complexes have been calculated using AutoDock 3.0.5. A linear relationship with correlation coefficient (R2) of 0.6 is obtained between ΔG(b)s values plotted against their corresponding IC50 values. The complex formed between Betv1 and the best docking pose for each ligand has been optimized by molecular dynamics simulation. Here, we describe the ligand binding of Betv1, which provides insight into the biological function of this protein. This knowledge is required for structural alteration or inhibition of some of these ligands in order to modify the allergenic properties of this protein.     

Miniaturized receptor binding assays: complications arising from ligand depletion

The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.     

De-intercalation of ethidium bromide and propidium iodine from DNA in the presence of caffeine

Caffeine (CAF) is capable of interacting directly with several genotoxic aromatic ligands by stacking aggregation. Formation of such hetero-complexes may diminish pharmacological activity of these ligands, which is often related to its direct interaction with DNA. To check these interactions we performed three independent series of spectroscopic titrations for each ligand (ethidium bromide, EB, and propidium iodine, PI) according to the following setup: DNA with ligand, ligand with CAF and DNA-ligand mixture with CAF. We analyzed DNA-ligand and ligand-CAF mixtures numerically using well known models: McGhee-von Hippel model for ligand-DNA interactions and thermodynamic-statistical model of mixed association of caffeine with aromatic ligands developed by Zdunek et al. (2000). Based on these models we calculated association constants and concentrations of mixture components using a novel method developed here. Results are in good agreement with parameters calculated in separate experiments and demonstrate de-intercalation of EB and PI molecules from DNA caused by CAF.     

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).     

The Bead blot: a method for selecting small molecule ligands for protein capture and purification

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.     

Circularly polarized luminescence studies of the ternary complexes formed by Tb(III) with (S,S)-ethylenediamine-N,N′-disuccinic acid and achiral substrate ligands

Circularly polarized luminescence spectroscopy has been used to study the ternary complexes formed by Tb(III) with (S,S)-ethylenediamine-N,N′-disuccinic acid (EDDS) and a series of achiral carboxylate ligands. The 1:1 Tb(EDDS) complexes form polynuclear species at low pH values, and only oxalic acid was able to interfere with this process. At elevated pH values the Tb(EDDS) compounds become monomeric, and are capable of forming ternary complexes. When the steric requirements of the substrate ligand were small, no perturbation of the EDDS stereochemistry was noted. However, certain strongly binding bidentate ligands with larger steric requirements were found to interact with the coordinated EDDS ligand. Evidence was also obtained which indicated that strongly binding terdentate ligands could partially displace one or more of the ligating carboxylates of the EDDS ligand.     

Syndecans play dual roles as cell adhesion receptors and docking receptors

Syndecan are a family of cell surface heparan sulfate proteoglycans that act as cell surface receptors. Most cell surface receptors have a limited number and type of ligand interactions, responding only to the binding of (a) specific ligand(s). In contrast, syndecans can interact with various numbers and types of ligands, and thus play more diverse roles than others. Various syndecan functions have not yet been fully classified and categorized, but we herein review previous studies suggesting that syndecans play dual function as cell surface receptors by acting as both adhesion receptors and docking receptors. Through this dual regulatory function, syndecans are capable of regulating both intra- and extracellular activities, potentially altering a variety of cell behaviors.     

Relationship between fructose 2,6-bisphosphate activation and MgATP inhibition of rat liver phosphofructokinase at high pH. Kinetic evidence for individual binding sites linked by finite couplings

The concentration of fructose 6-phosphate required to produce half-maximal velocity of rat liver phosphofructokinase at pH 9 (Ka) has been measured at 110 different combinations of MgATP and fructose 2,6-bisphosphate (Fru-2,6-BP) concentrations spanning the range 0.1-100 mM and 0.003-100 microM, respectively. The data have been evaluated by nonlinear regression to an equation resulting from a linked-function analysis of an enzyme capable of binding three ligands simultaneously at separate sites. In addition, the data have been fit to equations, derived from the linked-function expression, that would result if various combinations of antagonistic ligands were unable to bind to the enzyme simultaneously, even at high concentration, either because they compete for a single binding site or because they bind exclusively to different conformational forms of the enzyme. The complete linked-function equation is able to predict the Ka for rat liver phosphofructokinase as a function of any Fru-2,6-BP and/or MgATP concentration significantly better than any of the alternatives examined, particularly at high concentrations of one or both modifier ligands. The free energy couplings between all three possible pairs of ligands are of quite moderate magnitude, especially when the multiplicity of binding sites for each ligand that actually exists on the functional enzyme is considered. Therefore, we conclude that any explanation of the action of Fru-2,6-BP and MgATP by a model more elaborate than the simple linked-function case considered herein cannot be simplified by assuming that the properties of rat liver phosphofructokinase result from an equilibrium of limiting conformational states that exhibit exclusive binding properties.     

Analysis of substrate binding in individual active sites of bifunctional human ATIC

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.