Abstract
Characterization of the binding of a tumor necrosis factor (TNF) ligand to its receptor(s) is pivotal to understand how these proteins initiate signal transduction pathways. Unfortunately, kinetic elucidation of these interactions is strongly hampered by the multivalent nature of the binding partners. The interaction between TNF-related apoptosis-inducing ligand and its death receptors was analyzed using in-depth applications of surface plasmon resonance technology. Variations in receptor density and sensor chip type allowed us to manipulate the stoichiometry of the formed complex, and the rate constants describing the binding of trimeric TNF-related apoptosis-inducing ligand to only one receptor molecule were determined. Remarkably, the affinity of this trimer-monomer complex is in the picomolar range, and its dissociation very slow. Further analysis showed that the second and third receptor molecules bind with lower affinity to the preformed trimer-monomer complex. This together with results obtained with receptor activator of NF-κB ligand and B cell-activating factor strongly suggests that the binding of TNF family ligands to their receptors is initiated via the formation of a trimer-monomer complex that is sufficiently stable to allow binding of two additional receptor molecules. These results suggest that avidity does not play a significant role and thus provide new insight in how TNF ligands form the biologically important complexes with their receptors.
Highlights
Variations in receptor density and sensor chip type allowed us to manipulate the stoichiometry of the formed complex, and the rate constants describing the binding of trimeric tumor necrosis factor (TNF)-related apoptosisinducing ligand to only one receptor molecule were determined
Binding Cytokine to Captured Receptor—To capture receptor-Fc molecules to the surface of a sensor chip, protein A was directly immobilized to the chip surface of all flow cells in a Biacore 3000 instrument using a solution of 70 g/ml protein A in 10 mM NaAc, pH 4.5, and the primary amine coupling was performed according to the protocol of the supplier (GE Healthcare)
Complex Binding Behavior of recombinant human TRAIL (rhTRAIL) and Its Death Receptors at High Receptor Density—In many studies reported up to now, research groups have used an equilibrium approach to determine the affinity of the cytokine to its receptor
Summary
Unraveling the Binding Mechanism of Trivalent Tumor Necrosis Factor Ligands and Their Receptors Reis, Carlos R.; van Assen, Aart H. Despite good progress on structural insight, analysis of the interactions between TNF ligand family members and their receptors lacks unambiguous results This seems mainly caused by the multivalent character of these molecules, i.e. trimeric cytokines and often dimeric receptor-Fc fusions, which lead to complex kinetic behavior. Apart from presenting a method allowing unambiguous affinity determination, our results demonstrate that the binding mechanism of these cytokines is initiated via a high affinity interaction with the first receptor molecule, bringing the cytokine to the membrane
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