Ligand binding and receptor network formation in the tumor necrosis factor superfamily.

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a cytokine with anti-tumor potential, binds to transmembrane TRAIL receptors and initiates apoptosis. Although much has been characterized regarding intracellular signaling of TRAIL receptors, early events outside the cell and within the plasma membrane remain poorly understood. The central focus of this thesis is to establish biophysical interactions involved in ligand binding and subsequent receptor structural changes resulting in receptor activation. First, we demonstrate that TRAIL receptor 2 (death receptor 5, or DR5) forms receptor dimers in a ligand-dependent manner, and these receptor dimers exist within high molecular weight networks. We find that receptor dimerization relies upon covalent and non-covalent interactions between membrane-proximal residues, and that the transmembrane structure of two functional isoforms of DR5 are indistinguishable. This is the first evidence using endogenous, full-length receptor to demonstrate that DR5 networks are highly organized. Further, we show that DR5, upon stimulation by ligand, migrates into cholesterol rich membrane regions, and ligand-induced dimerization and network formation rely on cholesterol within the plasma membrane. Depletion of membrane cholesterol prevents structural changes associated with ligand binding as well as function. Therefore, lipid biophysical properties play an active role in determining receptor structure and function. Lastly, we identify and characterize a key, specific interaction between Methionine and aromatic residues that is critical for high affinity ligand-receptor binding and function in the TNF superfamily. Using structural bioinformatics, we demonstrate that this interaction--which occurs at approximately 5Å separation--is present in approximately one-third of known protein structures. Quantum calculations of model compounds and biological molecules demonstrate that this interaction provides additional stabilization over hydrophobic interactions and at distances out to 7Å, suggesting that this interaction may have evolved in proteins where a high degree of stabilization is required at longer distances. This motif may be utilized in the rational design of therapeutics targeting a range of proteins, including TNF members. In summary, our results characterize novel biophysical interactions between ligand-receptor, receptor-receptor, and receptor-membrane that together orchestrate a series of events that ultimately lead to cell death.