Interpretation of Scatchard plots for aggregating receptor systems.

Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function.     

Structural insight into the mechanism of activation of the Toll receptor by the dimeric ligand Spätzle

The Drosophila Toll receptor, which functions in both embryonic patterning and innate immunity to fungi and Gram-positive bacteria, is activated by a dimeric cytokine ligand, Sp?tzle (Spz). Previous studies have suggested that one Spz cross-links two Toll receptor molecules to form an activated complex. Here we report electron microscopy structures of the Toll ectodomain in the absence and presence of Spz. Contrary to expectations, Spz does not directly cross-link two Toll ectodomains. Instead, Spz binding at the N-terminal end of Toll predominantly induces the formation of a 2:2 complex, with two sites of interaction between the ectodomain chains, one located near to the N terminus of the solenoid and the other between the C-terminal juxtamembrane sequences. Moreover, Toll undergoes a ligand-induced conformational change, becoming more tightly curved than in the apo form. The unexpected 2:2 complex was confirmed by mass spectrometry under native conditions. These results suggest that activation of Toll is an allosteric mechanism induced by an end-on binding mode of its ligand Spz.     

Spatio-temporal dependence of the signaling response in immune-receptor trafficking networks regulated by cell density: a theoretical model

Cell signaling 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 signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a) how the perturbation caused by the signaling response propagates through the system; b) receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c) that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium ligand + surface receptor [Please see text] ligand - receptor complex. Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.     

Co-evolution of proteins with their interaction partners

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.     

Simulation of Association Curves and ‘Scatchard’ Plots of Binding Reactions where Ligand and Receptor are Degraded or Internalized

Abstract

Frequently during the course of binding to a receptor, ligand is degraded. In some preparations receptor is degraded. And with isolated cell preparations, ligand and/or receptor are internalized. Here we present a mathematical model for the combined binding and other reactions which gives useful information about the behaviour of such systems. The set of differential equations is solved numerically to simulate association curves and the resulting values of bound and free ligand are used to construct Scatchard plots. Where non-ideal conditions exist, the Scatchard plots are generally curvilinear. Dependence of this curvilinearity on time of measurement of free and bound ligand, on degradation and internalization of ligand and on degradation and internalization of receptor is shown. Equilibrium constants derived from the Scatchard plots are generally incorrect but the derived receptor concentration is often correct. The simulations lead to possibilities for distinction among the several side reactions in ligand-receptor binding systems.     

The theoretical analysis of kinetic behaviour of “hysteretic” allosteric enzymes V. Relaxation kinetics of dissociating enzyme systems

The expressions for relaxation time as a function of enzyme and specific ligand concentration are deduced for dissociating enzyme system 2p ? P (P is enzyme oligomer which is able to dissociate reversibly forming two identical halves p). It is assumed that ligand binding sites are equivalent and independent in each oligomeric enzyme form and the equilibrium between oligomeric forms develops rather slowly in comparison with the rate of the binding of the ligand. The kinetics of relaxation of the dissociating enzyme system 2p ? P with progressive change of the rate constants for association of oligomeric form p has been analysed in graphic form. The situations when one of the oligomeric enzyme forms is not able to bind the ligand are also considered. The principles of the analysis of relaxation kinetics of dissociating enzyme systems 2p ? P are discussed.     

Activation of transmembrane cell‐surface receptors via a common mechanism? The “rotation model”

It has long been thought that transmembrane cell‐surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand‐induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell‐surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding.     

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.     

Enthalpy-entropy compensation and cooperativity as thermodynamic epiphenomena of structural flexibility in ligand-receptor interactions

Ligand binding is a thermodynamically cooperative process in many biochemical systems characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to cooperativity of ligation needs to be elucidated. Here, we perform kinetic and thermodynamic analyses on a panel of cycle-mutated peptides, derived from influenza H3 HA(306-319), interacting with wild type and a mutant HLA-DR. We observe that, within a certain range of peptide affinity, this system shows isothermal entropy-enthalpy compensation (iEEC). The incremental increases in conformational entropy measured as disruptive mutations are added in the ligand or receptor are more than sufficient in magnitude to account for the experimentally observed lack of free-energy decrease cooperativity. Beyond this affinity range, compensation is not observed, and therefore, the ability of the residual interactions to form a stable complex decreases in an exponential fashion. Taken together, our results indicate that cooperativity and iEEC constitute the thermodynamic epiphenomena of the structural fluctuation that accompanies ligand-receptor complex formation in flexible systems. Therefore, ligand binding affinity prediction needs to consider how each source of binding energy contributes synergistically to the folding and kinetic stability of the complex in a process based on the trade-off between structural tightening and restraint of conformational mobility.     

The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor.

The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.     

Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. We have solved the x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations.     

The guanylyl cyclase receptor family

Cyclic GMP (cGMP) signals through protein kinases, ion channels, and possibly other effector systems as a second messenger. Its synthesis is regulated by guanylyl cyclase, whose activity is found in various cellular compartments including the plasma membrane and cytosol. A soluble form of guanylyl cyclase, which occurs as a heterodimer, appears to serve as a receptor for nitric oxide or nitrosothiols, or both. Recent research suggests the presence of multiple subtypes of the soluble form of guanylyl cyclase and tissue-specific expression of the different forms. At least two different forms of the plasma membrane guanylyl cyclase are known to occur in various mammalian tissues. One form, GC-A, is a receptor for atrial natriuretic peptide, and the binding of ligand causes marked increases in cGMP production. The other form, GC-B, is stimulated more effectively by a brain natriuretic peptide than by atrial natriuretic peptide, but its natural ligand remains in question. Both plasma membrane forms of the enzyme contain a single, putative transmembrane domain. The intracellular region of both forms contains a protein kinase-like domain just within the transmembrane domain. The protein kinase-like domain is followed by a cyclase catalytic region near the carboxyl terminus that is homologous to two internally homologous domains found in a bovine brain adenylyl cyclase. The possibility that other guanylyl cyclase receptor subtypes exist is now being explored. If they do, we may subsequently find that a diversity of specific ligands signals through cGMP.     

Simulation of Association Curves and ‘Scatchard’ Plots of Binding Reactions Where Ligand and Receptor are Degraded or Internalized

Abstract

Frequently during the course of binding to a receptor, ligand is degraded. In some preparations receptor is degraded. And with isolated cell preparations, ligand and/or receptor may be internalized. Here we present a mathematical model in which the binding and other reactions are combined. The resulting set of differential equations is solved numerically to simulate association curves and the resulting values of bound and free ligand are used to construct Scatchard plots. Where non-ideal conditions exist, the Scatchard plots are generally curvilinear. Dependence of this curvilinearity - on time of measurement of free and bound ligand, on degradation and internalization of ligand, and on degradation and internalization of receptor - is shown. Equilibrium constants derived from the Scatchard plots are generally incorrect but the derived receptor concentration is often correct. The simulations suggest experimental possibilities for distinction among the several side reactions in ligand-receptor binding systems.     

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.