Quantifying binding affinity. Quantify single cell-cell and cell-surface interactions under physiological conditions. This ground-breaking technique, known as single cell force spectroscopy SCFS , measures the interaction forces between a living cell attached to a cantilever and a target cell, functionalized substrate, tissue or biomaterial.
Read More. The life-time of a strong ligand-receptor interaction is much longer than the average time of ligand diffusion before the ligand can encounter with its binding partner. Moreover, the two-dimensional diffusions of receptors on plasma membrane are much slower than the three-dimensional diffusions of proteins in solvent environments.
As a result, the binding of different sites in a tethered ligand to their cell surface receptors becomes competitive. In another word, if one site of a ligand binds to its target receptor, it will take very long time for other unbound sites in the same ligand to find their target receptors, as the entire ligand-receptor complex diffuses on cell surfaces.
Meanwhile, the long dissociation time of the ligand-receptor complex, due to the strong affinity prevents other sites from diffusing back into the three-dimensional extracellular space and binding to their corresponding receptors. It needs to be noted that this kinetic trapping effect does not change the overall thermodynamics of the system. Therefore, when simulations reach infinite time, we should observe that most ligand sites can ultimately bind to their receptors due to the strong affinities.
However, the negative coupling due to the kinetic issue has more functional relevance in the context of understanding the role of spatial organization in multi-specific ligands, because these biological processes occur within the physiologically meaningful time scale.
It is reasonable to assume that both increase of encounter probability and decrease of overall dissociation of a multivalent complex are proportional to its internal structural flexibility, which has been validated by the further simulations. Our computational studies therefore provide quantitative insight into the general principles governing the binding between multivalent ligands and surface-bound receptors.
In the future, additional features will be integrated into the model for the application to specific biological systems. For instance, more specific information about structural fluctuations between different binding sites of a ligand and the binding constants of wild-type or mutated ligand-receptor interactions can be achieved by higher-resolution simulation methods such as Brownian dynamic simulation [ 45 — 54 ].
These data can be fed into the current rigid-body based model by the further development of a multi-scale framework.
Finally, it is worth mentioning that in some cases, binding of one ligand-receptor pair might change the affinity of other ligand-receptor pairs due to the conformational changes of these molecules upon binding. This effect is called allosteric regulation [ 55 ]. However, since molecules were simplified by rigid-bodies, the conformational changes within each ligand and receptor cannot be reflected by our model.
Therefore, the impacts of allosteric regulation on ligand-receptor interactions were not taken into account here. The principles revealed in this study are purely based on the spatial organization of multi-specific ligands. Future applications of our model include the design of multi-specific ligands to recognize specific cell types based on the differentiated expression levels of their surface receptors.
There exist large ranges of expression level for membrane receptors in different types of cells. For instance, expression of immune receptors on the surfaces of different T cells are highly variable, such that a wide spectrum of antigens can be targeted [ 56 ]. In cancer biology, specific mutations lead to the overexpression of certain receptors, such as cell adhesion molecules on membrane [ 57 ], which is a hallmark to distinguish tumor cells from normal cells [ 58 ].
Therefore, understanding the quantitative relation between ligand binding specificity and receptor expression level is important to maximize drug efficacy and minimize off-target drug toxicity. If a ligand is monomeric, its binding probability depends only on its concentration and the expression level of its target receptor.
Interestingly, by linking the ligand into a dimeric complex in which the second ligand subunit binds to a receptor with stable expression on cell surface, we show that the binding specificity of the first ligand not only depends on the expression level of its target receptor, but is also modulated by the binding affinity of the second ligand.
These results provide insights to the practical strategies of next-generation drug design. By generating multi-specific ligands with design principles based on binding affinity, topology of binding sites and expression levels of their cognate receptors, we will be able to control the selectivity of these ligands for specific cell types.
Conjugating these ligands with traditional cancer drugs may enable delivery to the target tissue with a much higher selectivity and reduced off-target effects [ 59 ].
Similarly, the incorporation of T cell receptor-specific recognition modules into tethered ligand assemblies may allow for the selective induction or suppression of disease-relevant T cells [ 60 ]. The selectivity associated with such reagents may reduce the extensive side effects associated with nearly all biologics-based immunotherapies, which elicit global immune modulation of the entire T cell repertoire [ 61 ].
The practical development of such ligand complexes could pave the way for a new generation of engineered immunotherapies. We thank Dr. Barry Honig for helpful discussions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
All relevant data are within the paper and its Supporting Information files. National Center for Biotechnology Information , U. PLoS Comput Biol. Published online Oct Steven C. Alexandre V Morozov, Editor. Author information Article notes Copyright and License information Disclaimer. Received Jun 14; Accepted Oct 2. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. Abstract The interactions between membrane receptors and extracellular ligands control cell-cell and cell-substrate adhesion, and environmental responsiveness by representing the initial steps of cell signaling pathways.
Author summary In order to adapt to surrounding environments, multiple signaling pathways have been evolved in cells. Introduction Integral membrane proteins are the sensors of extracellular signals, including cell-cell and cell-substrate interactions, as well as environmental queues.
Model and method We recently developed a rigid-body RB based model to simulate molecular binding in cellular environments [ 35 ]. Open in a separate window. Fig 1. Results Evaluate the relationship between affinity of individual binding sites and overall binding avidity in a multivalent complex We first investigated how the spatial organization of a multi-specific ligand affects binding between its individual binding sites and their receptors when their affinities are in different ranges.
Fig 2. To evaluate how spatial organization of a multi-specific ligand affects its binding with receptors, we fixed the binding affinity between receptor C and ligand D as -9kT. Fig 3. We systematically changed both AB binding affinity and CD binding affinity simultaneously.
Quantify the relation between binding affinities of ligands and their binding specificity to cells expressing different numbers of surface receptors The concentrations of receptors and ligands were fixed in the last section, with the surface density of receptor A equal to that of receptor C. Fig 4. We changed the relative concentrations of two receptors on cell surfaces. Fig 5. In order to investigate the functional role of binding site organization, four different topologies were designed.
Fig 6. The internal flexibility of multi-specific ligands was incorporated in the simulations. Discussion Binding of multivalent molecules is a ubiquitous phenomenon in living cells. PDF Click here for additional data file. Acknowledgments We thank Dr. Data Availability All relevant data are within the paper and its Supporting Information files. References 1. Packard B. Trends in Biochemical Sciences. Ullrich A, Schlessinger J. Cell-cell interactions in inflammation and cancer metastasis.
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For example, the three dimensional shape of the receptor protein is change upon the binding of the ligand. Also, the conformational state of a receptor protein will cause variations in the functional state of a receptor. Different types of ligands include substrates, inhibitors, activators, and neurotransmitters. Human cells use receptor-mediated endocytosis to take in cholesterol for use in the synthesis of membranes and as a precursor for the synthesis of other steroids.
Cholesterol travels in the blood in particles called low-density lipoproteins LDLs , complexes of lipids and proteins. These particles act as ligands hence they bind to LDL receptors on membranes and enter the cells by endocytosis. In humans with familial hypercholesterolemia, an inherited disease characterized by a very high level of cholesterol in the blood, the LDL receptor proteins are defective or missing so the LDL particles cannot enter cells.
Instead, cholesterol accumulates in the blood, where it contributes to early atherosclerosis. Atherosclerosis is the buildup of lipid deposits within the walls of blood vessels, causing of the bulge inwards of vessels and impeding blood flow. Note: These are the three types of endocytosis that the cell participates in. The third one represents the receptor-ligand binding mentioned for cholesterol in humans.
The binding of a ligand to a protein is greatly affected by the structure of the protein and is often accompanied by conformational changes. As an example, the specificity with which heme binds its various ligands changes when the heme is a component of myoglobin. When carbon monoxide binds to free heme molecules, it binds more than 20, times better than oxygen does, but it only binds times better than oxygen when the heme is bound in myoglobin.
The difference is most likely due to steric hindrance but there are other factors that have not yet been well-defined that may also affect the interaction of heme with carbon monoxide. Oxygen is poorly soluble in aqueous solutions and cannot be carried to tissues in sufficient quantity if it is only dissolved in blood serum.
The diffusion of oxygen through tissues is also ineffective over distances greater than a couple of millimeters. The evolution of larger, multicelluluar animals, though, depended on the evolution of proteins that could transport and store oxygen, but none of the amino acid side chains in proteins are suited for the reversible binding of oxygen molecules.
Ligand binding to receptors alters the chemical conformation , i. The conformational state of a receptor protein determines the functional state of a receptor. The tendency or strength of binding is called affinity. Ligands include substrates , inhibitors , activators , and neurotransmitters. Radioligands are radioisotope labeled compounds and used in vivo as tracers in PET studies and for in vitro binding studies.
The interaction of most ligands with their binding sites can be characterized in terms of a binding affinity. In general, high affinity ligand binding results from greater intermolecular force between the ligand and its receptor while low affinity ligand binding involves less intermolecular force between the ligand and its receptor. In general, high affinity binding involves a longer residence time for the ligand at its receptor binding site than is the case for low affinity binding.
High affinity binding of ligands to receptors is often physiologically important when some of the binding energy can be used to cause a conformational change in the receptor, resulting in altered behavior of an associated ion channel or enzyme.
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