Cytochrome P-450-dependent 14 alpha-demethylation of lanosterol in Candida albicans.

Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors.     

Evidence for a proteinase inhibitor binding component associated with murine spermatozoa

The supernatants of frozen-thawed murine epididymal sperm suspensions contain a heat-labile component capable of binding a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin. The substance has a molecular weight of approximately 15,000 and can be isolated by affinity chromatography using the inhibitor as the ligand. Although the substance has no inherent enzymatic properties, it will decrease the activity of the seminal inhibitor in the standard N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay. Enzyme-linked immunosorbent assays (ELISA) indicate that the substance, when bound to microtiter plates, is capable of binding the seminal vesicle inhibitor. Turkey egg white trypsin inhibitor will decrease the amount of the seminal inhibitor that will bind to the substance, while noninhibitor proteins, e.g., bovine serum albumin or insulin, have no effect. Turkey egg white and lima bean trypsin inhibitor will also decrease the amount of seminal vesicle inhibitor capable of binding to washed sperm. These data indicate the presence of an inhibitor acceptor site associated with murine epididymal spermatozoa.     

Differences between apo and three holo forms of the intestinal fatty acid binding protein seen by molecular dynamics computer calculations

It is commonly believed that binding affinity can be estimated by consideration of local changes of ligand and protein. This paper discusses a set of molecular dynamics simulations of intestinal fatty acid binding protein addressing the protein's response to presence or absence of different ligands. A 5-ns simulation was performed of the protein without a ligand, and three simulations (one 5-ns and two 2-ns) were performed with different fatty acids bound. The results indicate that, although the basic protein structure is unchanged by the presence of the ligand, other properties are significantly affected by ligand binding. For example, zero-time covariance patterns between protein, bound waters, and ligand vary between the different simulations. Moreover, the interaction energies between ligand and specific residues indicate that different ligands are stabilized in different ways. In sum, the results suggest that binding thermodynamics within this system will need to be calculated not from a subset of nearby protein:ligand interactions, but will depend on a knowledge of the motions coupling together water, protein, and ligand.     

Quantifying Escherichia coli glutaredoxin-3 substrate specificity using ligand-induced stability

Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.     

Capillary electrophoresis for the study of affinity interactions

Molecular recognition may be characterized both qualitatively and quantitatively by electrophoretic methods if complexed molecules differ in electrophoretic mobility from unbound ones. The use of capillary zone electrophoresis (CE) for the characterization of affinity interactions is advantageous because of the high resolution, reproducibility and wide applicability of the technique and because of the mild conditions, i.e., physiological buffers without additions of organics or detergents, that are often sufficient for highly efficient separations. CE gives the ability to characterize binding between small amounts of unlabelled reactants in solution, has few requirements for special characteristics of the interacting molecules and is also applicable to the study of interactions of individual components in mixtures, as detection of binding and analytical separation are achieved in one step. This is unique compared with other techniques for the study of non-covalent interactions. The advantages and disadvantages of using CE to demonstrate molecular interactions, to screen for specific ligand binding in complex mixtures and to calculate binding constants will be discussed.     

Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.     

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p‐Coumaric acid and related aromatic acids

Lignin comprises 15–25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP‐binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p‐coumarate, 3‐phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X‐ray crystal structures of protein–ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin‐derived aromatic compounds. The screens and structural data provide new functional assignments for these solute‐binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence‐based functional annotation methods for this family of proteins.Proteins 2013; 81:1709–1726. © 2013 Wiley Periodicals, Inc.     

Comparison of the effects of chemical and isotopic dilution on the dissociation of bound labeled ligands

The dissociation of a labeled ligand from a binding structure to which it is reversibly attached can be promoted either by dilution or by chase. The kinetics of the dissociation brought up by dilution can be modified or not by the presence of various concentrations of cold ligand, according to the molecular mechanism of interaction. Analog computer simulation leads to the following results: (i) no cooperativity, monomolecular dissociation: no modification; (ii) positive or negative cooperativity (sequential models): acceleration, no modification, or slowing down (according to the kinetic constants); (iii) positive cooperativity (concerted model): no modification; (iv) two-step interaction: no modification if both interaction steps take place in the same phase, otherwise acceleration; and, (v) bimolecular association and dissociation: acceleration. This methodology could be used in order to characterize the molecular properties of various binding structures in the field of drug and hormone receptors as well as in enzymology.     

Receptor binding competition: A paradigm for regulating TGF-β family action

The transforming growth factor (TGF)-β family is a group of structurally related, multifunctional growth factors, or ligands that are crucially involved in the development, regulation, and maintenance of animal tissues. In humans, the family counts over 33 members. These secreted ligands typically form multimeric complexes with two type I and two type II receptors to activate one of two distinct signal transduction branches. A striking feature of the family is its promiscuity, i.e., many ligands bind the same receptors and compete with each other for binding to these receptors. Although several explanations for this feature have been considered, its functional significance has remained puzzling. However, several recent reports have promoted the idea that ligand-receptor binding promiscuity and competition are critical features of the TGF-β family that provide an essential regulating function. Namely, they allow a cell to read and process multi-ligand inputs. This capability may be necessary for producing subtle, distinctive, or adaptive responses and, possibly, for facilitating developmental plasticity. Here, we review the molecular basis for ligand competition, with emphasis on molecular structures and binding affinities. We give an overview of methods that were used to establish experimentally ligand competition. Finally, we discuss how the concept of ligand competition may be fundamentally tied to human physiology, disease, and therapy.     

Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H' DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H' DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.     

Tet repressor mutants with altered effector binding and allostery

To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.     

Mathematical modelling and computational study of two-dimensional and three-dimensional dynamics of receptor–ligand interactions in signalling response mechanisms

Cell signalling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signalling pathways. But the binding process still presents many enigmas and, by analogy with surface catalytic reactions, two different mechanisms can be conceived: the first mechanism is related to the Eley–Rideal (ER) mechanism, i.e. the bulk-dissolved ligand interacts directly by pure three-dimensional (3D) diffusion with the specific surface receptor; the second mechanism is similar to the Langmuir–Hinshelwood (LH) process, i.e. 3D diffusion of the ligand to the cell surface followed by reversible ligand adsorption and subsequent two-dimensional (2D) surface diffusion to the receptor. A situation where both mechanisms simultaneously contribute to the signalling process could also occur. The aim of this paper is to perform a computational study of the behavior of the signalling response when these different mechanisms for ligand-receptor interactions are integrated into a model for signal transduction and ligand transport. To this end, partial differential equations have been used to develop spatio-temporal models that show trafficking dynamics of ligands, cell surface components, and intracellular signalling molecules through the different domains of the system. The mathematical modeling developed for these mechanisms has been applied to the study of two situations frequently found in cell systems: (a) dependence of the signal response on cell density; and (b) enhancement of the signalling response in a synaptic environment.     

Chemoreception by a cell with age-dependent receptor binding sites

The authors determine the time-dependent ligand current into a spherical cell that is covered with a large number of age-dependent receptors. These receptors can be in either of two states: active (i.e., available for ligand binding) or inactive. An active receptor turns inactive upon binding a ligand, and it can reappear as active at some later time. The transition inactive----active is treated as a probabilistic process. The ligand distribution around the cell is determined analytically in terms of this distribution at the cell surface. A set of nonlinear integral equations is derived for the distribution at the cell surface, which is solved numerically. In this way the time-dependent ligand current into the cell as well as the average active receptor population at the cell surface are determined.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

The Crystal Structure of Human Pyrin B30.2 Domain: Implications for Mutations Associated with Familial Mediterranean Fever

The inherited autoinflammatory syndrome familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, which are independent of any bacterial or viral infections. This disease is associated with point mutations in the mefv gene product pyrin. Although the precise molecular functions of pyrin are unknown, it seems to be involved in the maturation and secretion of interleukin-1β. Approximately two thirds of all FMF-associated mutations cluster in the C-terminal B30.2 domain of pyrin. To investigate the molecular consequences of FMF-associated mutations, we determined the crystal structure of the pyrin B30.2 domain at 1.35-Å resolution. The comparison with other B30.2/ligand complex structures revealed a shallow cavity, which seems to be involved in binding the pyrin ligand. The bottom of this cavity is covered mainly with hydrophobic amino acids, suggesting that pyrin recognizes its ligand by hydrophobic contacts and surface complementarities. FMF-associated mutations cluster around two sites on the B30.2 surface. Approximately two thirds, including those mutations with the most severe disease outcomes, are observed in the vicinity of the predicted peptide binding site, suggesting that they will have a direct impact on ligand binding. A second mutational hot spot was observed on the opposite side of the B30.2 domain in the neighbourhood of its artificial N-terminus. Although most FMF-associated mutations are solvent exposed, several will modify the main-chain conformation of loops. The experimental crystal structure of the pyrin B30.2 domain serves as a basis for an accurate modelling of these mutations.     

Curves of ligand binding. The use of hyperbolic functions for expressing titration curves

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/ radical2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK(1) and pK(2) for the first and second molecules of ligand are called pK*+/-logm, so that K*(2)=K(1)K(2) and m(2)=K(1)/K(2), then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*+/-log(1/2)[m+ radical(m(2)-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*+/-log(1/2)[m+ radical(m(2)-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m>/=2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.     

Cell surface receptors for signal transduction and ligand transport: a design principles study

Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor–ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i) avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii) consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii) dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.     

A Method for Removing Effects of Nonspecific Binding on the Distribution of Binding Stoichiometries: Application to Mass Spectroscopy Data

There is often an interest in knowing, for a given ligand concentration, how many protein molecules have one, two, three, etc. ligands bound in a specific manner. This is a question that cannot be addressed using conventional ensemble techniques. Here, a mathematical method is presented for separating specific from nonspecific binding in nonensemble studies. The method provides a way to determine the distribution of specific binding stoichiometries at any ligand concentration when using nonensemble (e.g., single-molecule) methods. The applicability of the method is demonstrated for ADP binding to creatine kinase using mass spectroscopy data. A major advantage of our method, which can be applied to any protein-ligand system, is that no previous information regarding the mechanism of ligand interaction is required.