The Structure of Tumor Necrosis Factor-α at 2.6 Å Resolution

Abstract

The three-dimensional structure of tumor necrosis factor (TNF-a), a protein hormone sgcreted by macrophages, has been determined at 2.6 A resolution by xray crystallography.Phases were determined by multiple isomorphous replacement using data collected from five heavy atom derivatives.The multiple isomorphous replacement phases were further improved by real space symmetry averaging, exploiting the noncrystallographic 3-fold symmetry of the TNF-a trimer.An atomic model corresponding to the known amino acid sequence of TNF-a was readily built into the electron density map calculated with these improved phases.The 17,350-dalton monomer forms an elongated, antiparallel @-pleated sheet sandwich with a "jelly-roll" topology.Three monomers associate intimately about a 3-fold axis of symmetry to form a compact bellshaped trimer.Examination of the model and comparison to known protein structures reveals striking structural homology to several viral coat proteins, particularly satellite tobacco necrosis virus.Locations of residues conserved between TNF-a and lymphotoxin (TNF-@, a related cytokine known to bind to the same receptors as TNF-a) suggest that lymphotoxin, like TNF-a, binds to the receptor as a trimer and that the general site of interaction with the receptor is at the "base" of the trimer.Tumor necrosis factor-a (TNF-a)' is a pluripotent cytokine secreted by macrophages in response to a variety of inflammatory agents, particularly bacterial endotoxin.TNF-a was also isolated as cachectin (1, 2) which has been shown to mediate endotoxin-induced fever (3), shock (4, 5), and cachexia (wasting) ( 6), (and is believed to play a role in the cachectic state induced by many chronic diseases including cancer and AIDS.Th.e cytokine also induces hemorrhagic necrosis of certain transplantable tumors in mice and is specifically cytotoxic to a variety of tumor cells in culture (7, 8).The biological functions of the molecule are mediated by high affinity binding to receptors expressed on many cell types; specific effects of receptor binding include inhibition of triglyceride metabolism in adipocytes (1, 9-11), activation of neutrophils (12,13), and stimulation of proto-oncogenes cmyc and c-fos in fibroblasts (14).TNF-a is encoded in the