Thermodynamics and kinetics of co-operative protein-nucleic acid binding: II. Studies on the binding between protamine and calf thymus DNA

Unspecific binding of a protamine, namely fluorescein-labelled clupeine Z, to double-stranded calf thymus DNA was studied using fluorescence titration methods and chemical relaxation techniques. Both equilibrium and kinetic data have been analysed using general theoretical approaches discussed in the accompanying paper. The results agree well with the predictions made on the basis of a standard co-operative binding model.Basic parameters evaluated are the co-operative binding constant (K), the coefficient measuring co-operative interaction between nearest neighbours (q), the number of nucleotides occupied by one protamine molecule (n) and the rate constant of dissociation at the ends of bound ligand sequences (K_D). Values obtained at 20 °C, pH 7.5 and 0.4 m-NaCl were K = 5.8 × 10⁷m^?1, q = 1700, n = 20 and K_D = 0.29 s^?1. They have been found to be sensitive to the concentration of added salt (NaCl). This effect apparently reflects the essentially electrostatic nature of the binding process. The results can be satisfactorily described in terms of competitive binding of sodium ions.     

The molecular basis of co-operative DNA binding between lambda integrase and excisionase

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.     

Thermodynamics and kinetics of co-operative protein-nucleic acid binding: I. General aspects of analysis of data

The process under consideration is the binding of a ligand to a linear polymer of equivalent subunits such that each bound molecule of ligand occupies n subunits. Interactions between bound ligand molecules are also considered. Some useful points regarding the evaluation of raw data without recourse to any specific binding mechanism are discussed first. For a treatment in terms of appropriate thermodynamic parameters a simple model is examined in greater detail. It assumes that interactions are limited to those between ligands bound to nearest-neighbour positions on the polymer.Exact expressions for some basic binding properties of this model at equilibrium are developed. The relations can be considerably simplified in the case of pronounced positive co-operativity which is frequently encountered in practice. Appropriate plots of the data to test the model and to evaluate its parameters are proposed.A simple but consistent kinetic scheme is also introduced. It allows calculation of relaxation times as they can be measured by means of special techniques.     

On the interpretation of binding isotherms in complex biological systems: Apparent homogeneity of some heterogeneous systems

A simple treatment of the effect of site heterogeneity upon binding isotherms is presented, which is applicable to the analysis of data obtained from measurements of hormone, drug, or lectin binding to membranes and cell surfaces. Using this treatment, isotherms corresponding to various distributions of binding constants have been fitted to examples of experimental binding data ordinarily interpreted in the context of a homogeneous binding site model. It is found that these data do not permit one to exclude the alternate possibility of a broad distribution of the binding constant $\text{K}$. If a homogeneous binding site model can be satisfactorily fitted to the data, it is probable that the value of $\text{K}$ obtained by this procedure is equal or nearly equal to the number average value of $\text{K}$ in the actual (unknown) distribution.     

On the interpretation of binding isotherms in complex biological systems. Apparent homogeneity of some heterogeneous systems

A simple treatment of the effect of site heterogeneity upon binding isotherms is presented, which is applicable to the analysis of data obtained from measurements of hormone, drug, or lectin binding to membranes and cell surfaces. Using this treatment, isotherms corresponding to various distributions of binding constants have been fitted to examples of experimental binding data ordinarily interpreted in the context of a homogeneous binding site model. It is found that these data do not permit one to exclude the alternate possibility of a broad distribution of the binding constant K. If a homogeneous binding site model can be satisfactorily fitted to the data, it is probable that the value of K obtained by this procedure is equal or nearly equal to the number average value of K in the actual (unknown) distribution.     

Free energy coupling within macromolecules. The chemical work of ligand binding at the individual sites in co-operative systems

Individual-site binding curves such as those obtainable from techniques of DNase footprinting or nuclear magnetic resonance spectroscopy can be used to monitor structurally localized events within biopolymers. This paper discusses thermodynamic aspects of individual-site ligand binding for co-operative systems where the binding of ligand at a local site is coupled to binding of the same ligand species at other sites within the macromolecule. Individual-site binding isotherms have the following properties. (1) They provide a direct indication of the role played by the particular site in the overall binding reaction. (2) They can be used to determine the energetic contribution of loading the site regardless of the complexity of the system. (3) They can be used to resolve microscopic equilibrium constants and co-operativity constants in cases where the classical isotherm is incapable of such resolution. The microscopic constants bear a complex relation to the chemical work of loading each individual site. For a system with two interacting sites we derive analytical relationships between the individual-site loading energies and the microscopic constants. These relationships prescribe, for any values of the microscopic constants, how the co-operative energy is partitioned between events at the two sites. At fixed ligand activity the binding free energy can be estimated directly from an individual-site isotherm. This quantity, which is also a composite of the microscopic constants, provides a useful measure of site--site interaction. Several examples and applications are discussed for these properties of individual-site binding reactions.     

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane-actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.     

Molecular mechanisms and kinetics between DNA and DNA binding ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.     

Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics

The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies.     

Genetic selection for mutations that impair the co-operative binding of lambda repressor

Bacteriophage λ repressor binds co-operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant λ repressors that have lost the ability to bind co-operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co-operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl-terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein-DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.     

Parameter estimation for ligand binding systems kinetics applied to 1,25-dihydroxycholecalciferol

A mathematical analysis of the kinetics of the hormone-receptor interaction was applied to the 1,25-dihydroxycholecalciferol-intestinal receptor system. The exact analytical solution and the numerical integration of the kinetic equation were installed in a Statistical Analysis System (SAS) computer program to estimate the rate constants of the reaction. Estimates of the parameters obtained by these two methods are similar, demonstrating that the numerical integration can be combined with the nonlinear regression procedure for least-squares parameter fitting using a simple SAS program. This enables estimation of kinetics rate constants when the kinetic equation cannot be solved analytically. The ratio of the rate constants (ka/kd) found by the nonlinear procedure is close to the independently determined equilibrium (Scatchard) constant in the nonlinear analysis.     

Bivalent and co-operative binding of complement factor H to heparan sulfate and heparin

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.     

Effects of co-operative ligand binding on protein amide NH hydrogen exchange

Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in the ligand binding.     

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits.Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path.The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.     

'Multimodal' kinetics: Cyanobacterial nitrate reductase and other enzyme, transport and binding systems

Concentration‐dependence data for nitrate reductase (EC 1.7.99.4) from heterocystous, nitrogen‐fixing cyanobacteria (J. Martín‐Nieto, E. Flores and A. Herrero, 1992. Plant Physiol. 100: 157–163) have been interpreted most plausibly to reflect the operation of a single enzyme with two independent catalytic sites. However, data from a total of 30 experiments (published as well as unpublished) are, overall, much better ( P < 0.0001) represented according to a 'multimodal' kinetic model rather than as due to two separate sites. This new term is introduced to refer to enzyme systems displaying multiple concentration‐dependent phases separated by sharp inflections. This phenomenon is taken to reflect the operation of a single catalytic site undergoing discontinuous conformational transitions and thus able to function in distinct kinetic 'modes'. Moreover, plots of log K_m versus log V_max in these kinetic systems are perfectly linear, as also previously found for multiphasic plant uptake systems. The same multi‐mode kinetic behavior is exhibited by a wide variety of enzyme, uptake and ligand‐binding systems from plants, animals and microorganisms, including monomeric proteins purified to homogeneity. Multimodal kinetics thus constitute a widespread, albeit largely unrecognized, phenomenon in nature.     

Quadruplex-coupled kinetics distinguishes ligand binding between G4 DNA motifs

G-quadruplex (or G4 DNA) specific ligands are important potential anticancer molecules as telomerase inhibitors. On the other hand, emerging evidence implicates G4 DNA in regulation of several oncogenes making telomerase inhibitors amenable to undesired effects (Borman, S. (2007) Chem. Eng. News 85 (22), 12-17). Therefore molecules which can discriminate between G4 DNA are of interest, both as telomerase inhibitors and for selective intervention of gene expression. Design of selective molecules requires resolution of the coupled equilibria between intramolecular quadruplex-formation and bimolecular ligand-binding. Several previous studies have reported G4-ligand binding kinetics, however the primary equilibrium of intramolecular G4 DNA folding/unfolding was not considered. Here, we quantitatively assess the linked equilibrium in G4-ligand complexes using a novel real time surface plasmon resonance-based technique. Kinetic constants for G4 folding/unfolding and ligand binding were simultaneously determined, for the first time, from a single reaction by resolving the coupled equilibrium. We demonstrate the coupled model by showing that affinity of TMPyP4 (a well-established anticancer telomerase inhibitor) for the human telomere quadruplex is only 3-fold more than the c-MYC promoter G4, which is known to repress c-MYC. This provides quantitative rationale to poor selectivity of TMPyP4 in recently observed cell-based assays. In the light of recent advances indicating G4's regulatory potential in several important genes, quantitative evaluation of selectivity vis-à-vis affinity as presented here will augment design and preliminary screening of new molecules.     

Determination of binding strength and kinetics of binding initiation

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5×10⁻³ dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1–10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

A quantitative plot for the determination of negatively co-operative ligand binding sites in proteins

The paper describes a method for determining the number of interacting ligand binding sites for co-operative proteins. The method is simple and is particularly applicable in the case of negatively co-operative ligand binding.     

Interplay between binding affinity and kinetics in protein–protein interactions

To clarify the interplay between the binding affinity and kinetics of protein–protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound‐state valley is deep with a barrier height of 12 ? 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920–933. © 2016 Wiley Periodicals, Inc.     

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.