Differences in electrostatic properties at antibody-antigen binding sites: implications for specificity and cross-reactivity

Antibodies HyHEL8, HyHEL10, and HyHEL26 (HH8, HH10, and HH26, respectively) recognize highly overlapping epitopes on hen egg-white lysozyme (HEL) with similar affinities, but with different specificities. HH8 binding to HEL is least sensitive toward mutations in the epitope and thus is most cross-reactive, HH26 is most sensitive, whereas the sensitivity of HH10 lies in between HH8 and HH26. Here we have investigated intra- and intermolecular interactions in three antibody-protein complexes: theoretical models of HH8-HEL and HH26-HEL complexes, and the x-ray crystal structure of HH10-HEL complex. Our results show that HH8-HEL has the lowest number and HH26-HEL has the highest number of intra- and intermolecular hydrogen bonds. The number of salt bridges is lowest in HH8-HEL and highest in HH26-HEL. The binding site salt bridges in HH8-HEL are not networked, and are weak, whereas, in HH26-HEL, an intramolecular salt-bridge triad at the binding site is networked to an intermolecular triad to form a pentad. The pentad and each salt bridge of this pentad are exceptionally stabilizing. The number of binding-site salt bridges and their strengths are intermediate in HH10-HEL, with an intramolecular triad. Our further calculations show that the electrostatic component contributes the most to binding energy of HH26-HEL, whereas the hydrophobic component contributes the most in the case of HH8-HEL. A "hot-spot" epitope residue Lys-97 forms an intermolecular salt bridge in HH8-HEL, and participates in the intermolecular pentad in the HH26-HEL complex. Mutant modeling and surface plasmon resonance (SPR) studies show that this hot-spot epitope residue contributes significantly more to the binding than an adjacent epitope residue, Lys-96, which does not form a salt bridge in any of the three HH-HEL complexes. Furthermore, the effect of mutating Lys-97 is most severe in HH26-HEL. Lys-96, being a charged residue, also contributes the most in HH26-HEL among the three complexes. The SPR results on these mutants also highlight that the apparent "electrostatic steering" on net on rates actually act at post-collision level stabilization of the complex. The significance of this work is the observed variations in electrostatic interactions among the three complexes. Our work demonstrates that higher electrostatics, both as a number of short-range electrostatic interactions and their contributions, leads to higher binding specificity. Strong salt bridges, their networking, and electrostatically driven binding, limit flexibilities through geometric constrains. In contrast, hydrophobic driven binding and low levels of electrostatic interactions are associated with conformational flexibility and cross-reactivity.     

Ligand competition curves as a diagnostic tool for delineating the nature of site-site interactions: theory

A few molecular models have been developed in recent years to explain the mechanism of cooperative ligand binding. The concerted model of Monod, Wyman and Changeux and the sequential model of Koshland, Némethy and Filmer were formulated to account for positively cooperative binding. The pre-existent asymmetry model and the sequential model can account for negatively cooperative ligand binding. In most cases, however, it is virtually impossible to deduce the molecular mechanism of ligand binding solely from the shape of the binding isotherm. In the present study we suggest a new strategy for delineating the molecular mechanism responsible for cooperative ligand binding from binding isotherms. In this approach one examines the effect of one ligand on the cooperativity observed in the binding of another ligand, where the two ligands compete for the same set of binding sites. It is demonstrated that the cooperativity of ligand binding can be modulated when a competitive ligand is present in the protein-ligand binding mixture. A general mathematical formulation of this modulation is presented in thermodynamic terms, using model-independent parameters. The relation between the Hill coefficient at 50% ligand saturation with respect to ligand X in the absence, h(x), and in the presence of a competing ligand Z, h(x,z), is expressed in terms of the thermodynamic parameters characterizing the binding of the two ligands. Then the relationship between h(x) and h(x,z), in terms of the molecular parameters of the different allosteric models, is explored. This analysis reveals that the different allosteric models predict different relationships between h(x,z) and h(x). These differences are especially focused when Z binds non-cooperatively. Thus, it becomes possible, on the basis of ligand binding experiments alone, to decide which of the allosteric models best fits a set of experimental data.     

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

Disparate degrees of hypervariable loop flexibility control T-cell receptor cross-reactivity, specificity, and binding mechanism

αβ T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the αβ TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3α and CDR3β hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3β possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3α is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.     

Statistical analysis of drug-drug and site-site interactions with isobolograms

The use of more than one drug to achieve a desired effect has been a common practice in pharmacologic testing and in clinical practice. For example, combinations of analgesics are frequently prescribed with a view to enhancing pain relief and reducing adverse effects. It is also well established that administration of more than one drug may give effects that are greater than, or less than, the additive effect of each drug given individually. A non-mechanistic method of characterizing the effect resulting from the administration of two compounds is the isobologram. It is relatively simple to draw and interpret isobolograms. However, this graphical technique, which employs equieffective concentrations of individual drugs and combinations of these, obtains the concentrations as random variables from concentration-effect data, usually transformed to a parallel line assay. Thus, statistical confidence limits from such assays, as well as from non-parallel designs, must be expressed on the isobologram if this diagram is to establish superadditive, subadditive, or merely additive effects. We now present a detailed statistical analysis of the isobolographic method illustrated with examples of the statistical procedures, a rational basis for selecting proportions of each drug in the combination, and a relatively novel application of the isobolographic concept, i.e., interactions involving different anatomical sites.     

Some equilibrium and non-equilibrium properties of the allosteric interactions in enzymes. I. Ligand binding and saturation equations

Recent evidence and speculation regarding the dynamic structure of biological membranes is combined with information on the pharmacology and biochemistry of the allergic histamine release reaction to formulate a model which can explain many of the observed events in this reaction, and especially tie presumed early enzymological events to pharmacologically controlled subsequent events. It is proposed that the reaction of the cell-bound antibodies with suitable antigens causes a membrane deformation or a displacement of hydrophilic residues within the membrane in such a manner that there is a local clustering or polarization of charges. Biochemically, the earliest event may be the activation of a membrane-bound chymotrypsin-like proesterase. A speculative step in the proposed sequence is the activation of a membrane-bound pro-phospholipase A by the activated esterase. The local, limited action of the phospholipase A on the membrane lipids could influence the action of membrane-bound ATPase and nucleotide cyclases in several ways. The resulting local decrease in the concentration of cyclic AMP, is a condition which is known to modulate antigen-induced histamine release. It is proposed that the cyclic nucleotides may affect histamine release at more than one point in the sequence. First, they may regulate the contractility of the microtubles which have been shown to be involved in histamine release. Second, they may influence the state of aggregation and subcellular distribution of the microfilaments which play a role in the maintenance of the normal organization of the cell. As a result of the drop in the cyclic AMP concentration, or the accumulation of lysophosphatides, the cell membrane may be reorganized. This could lead to membrane invagination and an apparent “interiorization” of some of the aqueous milieu. The histamine-containing granules of the mast cells are thus brought into proximity of these deep invaginations by microtubule action, an energy-requiring process. The perigranular membranes fuse with the plasma membrane and the granules exchange their stored histamine for the extracellular sodium which enters the invaginations with the water. The histamine is then equilibrated with the external medium. A number of alternative mechanisms and testable corollaries of the theory are discussed.     

Site-directed alkylation and site-site interactions in bovine seminal ribonuclease

Active-site histidine residues of bovine seminal RNase have been found to react with bromoacetic acid and with 2'(3')-O-bromoacetyluridine (BrAcUrd) at a much faster rate than free histidine. The former reagent reacts preferentially at the pros-N of His119, the latter is specific for the tele-N of His12. Alkylation with bromoacetic acid is mutually exclusive for either His119 or His12 and takes place predominantly at His119, while with BrAcUrd alkylation was found to be selective for His12. These results are very similar to those obtained with the same reagents on RNase A, confirming that seminal and pancreatic ribonucleases have similar geometries at their active sites. On the other hand, the kinetics of reaction of bromoacetyluridine with seminal RNase reveal a 'half-of-the sites' reactivity of the enzyme for this reagent, which is found to discriminate between the two structurally identical active sites of the dimeric enzyme.     

Ligand binding specificity of a neutral L-amino acid olfactory receptor

1. The ligand binding specificity of the L-[3H]alanine binding site was investigated in isolated cilia preparations from the olfactory epithelium of channel catfish (Ictalurus punctatus) by competitive binding experiments. 2. Approximately 45 amino acids, derivatives and enantiomers were tested for the ability to compete with radiolabeled L-alanine for common binding sites. 3. Acidic and basic L-amino acids and imino acids did not compete as effectively as L-alanine for the receptor, while long-chain neutral ligands were only partially effective inhibitors of L-alanine binding. 4. D-Alanine and L-alanine derivatives with substituted alpha-amino or carboxyl groups exhibited decreased ability to compete for the receptor, paralleling their lower neurophysiological potency. 5. In combination, the ligand binding results were consistent with previous electrophysiological data in catfish, and suggest the presence of an olfactory receptor site that selectively recognizes short-chain neutral amino acids.     

Cooperativity in RNA-protein interactions: global analysis of RNA binding specificity

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach,we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.     

Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.     

The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.     

Differences between non-specific and bio-specific, and between equilibrium and non-equilibrium, interactions in biological systems

The interaction forces between biological molecules and surfaces are much more complex than those between non-biological molecules or surfaces, such as colloidal particle surfaces. This complexity is due to a number of factors: (i) the simultaneous involvement of many different molecules and different non-covalent forces - van der Waals, electrostatic, solvation (hydration, hydrophobic), steric, entropic and 'specific', and (ii) the flexibility of biological macromolecules and fluidity of membranes. Biological interactions are better thought of as 'processes' that evolve in space and time and, under physiological conditions, involve a continuous input of energy. Such systems are, therefore, not at thermodynamic equilibrium, or even tending towards equilibrium. Recent surface forces apparatus (SFA) and atomic force microscopy (AFM) measurements on supported model membrane systems (protein-containing lipid bilayers) illustrate these effects. It is suggested that the major theoretical challenge is to establish manageable theories or models that can describe the spatial and time evolution of systems consisting of different molecules subject to certain starting conditions or energy inputs.     

Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions

A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.     

Ligand binding: functional site location,similarity and docking

Computational methods for the detection and characterisation of protein ligand-binding sites have increasingly become an area of interest now that large amounts of protein structural information are becoming available prior to any knowledge of protein function. There have been particularly interesting recent developments in the following areas: first, functional site detection, whereby protein evolutionary information has been used to locate binding sites on the protein surface; second, functional site similarity, whereby structural similarity and three-dimensional templates can be used to compare and classify and potentially locate new binding sites; and third, ligand docking, which is being used to find and validate functional sites, in addition to having more conventional uses in small-molecule lead discovery.     

Ligand binding and detoxication

In an organism the binding of a toxic chemical to a binding site can act as a detoxication mechanism when toxicity is a property of the unbound ligand. This qualitative statement has been evaluated in quantitative terms. To this end parameters have been defined for which numerical values are required, equations are derived and a procedure is outlined that allows assessment of when and to what extent binding is of value in detoxication. In the process two new quantities are introduced, i.e. the binding capacity and the binding activity, which make for easier handling and comparison of binding data. It is concluded that to be important in detoxication the numerical value of the binding activity must be greater than unity and the total ligand concentration should not exceed the binding capacity. These general conclusions can be further refined depending on the accuracy with which the values of the parameters involved are known. Due to its generality the results of the analysis are useful in all situations where it is desirable to know the magnitude of the free fraction of a bound chemical.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

Ligand binding specificity of the leukocyte response integrin expressed by human neutrophils.

The ligand binding specificity of the leukocyte response integrin (LRI) expressed by polymorphonuclear leukocyte (PMN) was investigated by examining its interaction with two adhesion motifs within fibrinogen: the alpha chain sequence RGD and the gamma chain sequence KQAGDV. The effect of the hexapeptides KQAGDV, KQRGDV, and KGAGDV on fibrinogen-stimulated phagocytosis, a LRI-dependent function, was examined. Surprisingly, the sequence KGAGDV was most potent for inhibition of fibrinogen-stimulated ingestion; the order of potency of these peptides was KGAGDV greater than KQAGDV greater KQRGDV = GRGDSPA. Latex spheres coated with multivalent KGAGDV bound specifically to PMN and antibodies that recognized either the LRI beta chain (7G2) or an associated protein (IAP)-abrogated bead binding. Various control and anti-beta 1 and anti-beta 2 antibodies did not affect bead binding. Monovalent peptides KGAGDV and KQRGDV were equipotent for inhibition of bead binding to unstimulated PMN (ID50 = 19 microM). In contrast, KGAGDV was more potent than KQRGDV for inhibition of bead binding to N-formylmethionylleucylphenylalanine-stimulated PMN (ID50 = 2.5 microM versus ID50 = 60 microM). A control peptide, KGALEVA, did not inhibit LRI ligand binding or function. These data suggest that the unique amino acid sequence KGAGDV may represent a specific ligand for LRI and that LRI ligand binding specificity may be regulated by the activation state of the cell.