Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis.

Abstract

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32‐‐‐‐Trp, Leu36‐‐‐‐Phe, Ser86‐‐‐‐Phe and Ala84‐‐‐‐Val) behaved similar to the wild‐type in various physico‐chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84‐‐‐‐Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non‐conservative alterations were introduced (Leu29‐‐‐‐Ser and Glu146‐‐‐‐Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val‐‐‐‐Ala) caused a significant drop (500‐fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor‐interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.

Kinetic Analysis of Adenovirus Fiber Binding to Its Receptor Reveals an Avidity Mechanism for Trimeric Receptor-Ligand Interactions

“…The interactions of the TNF receptor with TNF␤ (29) and TRAIL (TNF-related apoptosis inducing ligand) with DR5 (death receptor 5) (30) are surprisingly similar to that of the CAR D1 domain with adenovirus type 12 fiber head (11), with the receptor binding in a groove between two monomers and surface loops located toward the bottom of the trimer being functionally most relevant (10,11,31,32). It is therefore likely that the same avidity mechanism we observe for adenovirus fiber head binding to CAR also exists for ligands binding to the TNF receptor family.…”

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

“…4). These intersubunit grooves are the main site for interaction between TNF and TNF-R as clear from mutagenesis studies (8) and crystallography of human lymphotoxin bound to hTNF-RI (7). Furthermore, amino acids 32 and 86 of hTNF, which are crucial for binding to hTNF-RI, are also located near these intersubunit grooves (8 -10).…”

Humanization and Epitope Mapping of Neutralizing Anti-Human Fas Ligand Monoclonal Antibodies: Structural Insights into Fas/Fas Ligand Interaction

“…In the lower contact region, Y218 on the FasL D-E loop that is conserved among death-inducing TNF family members (Fig. 5A) has been reported to be important for LT-␣ and TNF-␣ binding to TNFRI (19,29,30). In addition, R86 on the second CRD of Fas has been shown to be a critical residue for direct interaction with FasL Y218 (31).…”