The structure of mouse tumour-necrosis factor at 1.4 Å resolution: towards modulation of its selectivity and trimerization

Abstract

The 1.4 A resolution structure of recombinant mouse tumour-necrosis factor alpha (mTNF) at 100 K has been determined. The crystals are triclinic, space group P1, with unit-cell parameters a = 48.06, b = 48.18, c = 51.01 A, alpha = 114.8, beta = 103.6, gamma = 91.1 degrees. The structure was refined to a final crystallographic R value of 19.7% (Rfree = 23.3%), including 3477 protein atoms, one 2-propanol molecule, one Tris molecule and 240 water molecules. Throughout the crystal lattice, the trimers are differently packed compared with human TNF, which was crystallized in the tetragonal space group P41212 and refined to 2.6 A resolution. The structures of mTNF and human TNF are very similar, diverging mainly in regions that are either flexible and/or involved in crystal packing. Some loops in mTNF which contain residues important for receptor binding are better resolved than in human TNF, such as the surface-exposed loops 30-34 and 144-147, which are also important for receptor specificity. Compared with human TNFs, the channel formed by the three monomers in mTNF is narrower. One 2-propanol molecule trapped in the trimeric channel could be a lead compound for the design of TNF inhibitors.

Identification of Tumor Necrosis Factor (TNF) Amino Acids Crucial for Binding to the Murine p75 TNF Receptor and Construction of Receptor-selective Mutants

“…The three-dimensional structure of mTNF (20) shows that amino acids 102-104, which differ in mTNF and hTNF, are located close to amino acids 71-73 (Fig. 4).…”

“…Although the structure of hTNF and mTNF is very similar (20,28), the amino acids 71-73, 89, and 102 are all located in flexible, surface-exposed loops that differ strongly (loops 64 -76, 84 -91, and 99 -112). Moreover, the amino acids 71-73, 89, and 102 are present near the intersubunit grooves of the TNF trimer (Fig.…”

Immunochemical termination of self-tolerance

“…mTNF-␣ was chosen as the target protein for this study because: (i) it is a well characterized cytokine involved in the regulation of infectious, inflammatory, and autoimmune phenomena (12); (ii) the biological properties of this protein have been extensively studied including its structure, function, and signaling mechanisms (12)(13)(14)(15)(16); and (iii) mTNF-␣ knockout mice are viable and show no apparent phenotypic abnormalities (15), suggesting that mice will survive a neutralizing immune response against TNF-␣. In addition, anti-TNF-␣ antibodies (17,18) and soluble chimeric TNF-␣ receptors (19,20) are widely used in the treatment of autoimmune disease, and a number of approaches are being pursued to develop TNF-␣-specific vaccines for clinical use.…”

PanR1, a Dominant Negative Missense Allele of the Gene Encoding TNF-α (Tnf), Does Not Impair Lymphoid Development

“…2C) in close proximity to residues that have been shown to interact with the Tnfrsf1a receptor (region IV) ( Fig. 2D) (37,(41)(42)(43).…”

“…The crystal structure of Lta (which exhibits a conformation similar to that of TNF (47)) bound to Tnfrsf1a, as well as the study of point mutations in Tnf, has defined four main regions involved in ligand-receptor interaction (regions I to IV) (37,(41)(42)(43). P138, and its equivalent amino acid in Lta (I154), are both located in the tight turn preceding region IV.…”