Mutations in the binding site of TNFR1 PLAD reduce homologous interactions but can enhance antagonism of wild-type TNFR1 activity.

The tumour necrosis factor receptor superfamily (TNFRSF) members contain cysteine-rich domains (CRD) in their extracellular regions, and the membrane-distal CRD1 forms homologous interactions in the absence of ligand. The CRD1 is therefore termed a pre-ligand assembly domain (PLAD). The role of PLAD-PLAD interactions in the induction of signalling as a consequence of TNF-TNFR binding led to the development of soluble PLAD domains as antagonists of TNFR activation. In the present study, we generated recombinant wild-type (WT) PLAD of TNFR1 and mutant forms with single alanine substitutions of amino acid residues thought to be critical for the formation of homologous dimers and/or trimers of PLAD (K19A, T31A, D49A and D52A). These mutated PLADs were compared with WT PLAD as antagonists of TNF-induced apoptosis or the activation of inflammatory signalling pathways. Unlike WT PLAD, the mutated PLADs showed little or no homologous interactions, confirming the importance of particular amino acids as contact residues in the PLAD binding region. However, as with WT PLAD, the mutated PLADs functioned as antagonists of TNF-induced TNFR1 activity leading to induction of cell death or NF-κB signalling. Indeed, some of the mutant PLADs, and K19A PLAD in particular, showed enhanced antagonistic activity compared with WT PLAD. This is consistent with the reduced formation of homologous multimers by these PLAD mutants effectively increasing the concentration of PLAD available to bind and antagonize WT TNFR1 when compared to WT PLAD acting as an antagonist. This may have implications for the development of antagonistic PLADs as therapeutic agents.