Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55 ) mediates a multitude of effects both in normal and in tumor cells. Cluste ring of the intracellular domain of the receptor, the so-called death domai n (DD), is responsible for both the initiation of cell killing and the acti vation of gene expression. To characterize this domain further, TNFR55 DD w as expressed and purified as a thioredoxin fusion protein in Escherichia co li. Circular dichroism, steady-state and time-resolved fluorescence spectroscop y were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas -associating protein with DD (FADD) and p75 nerve growth factor receptor, f or which the 3-dimensional structure are already known. The structural info rmation derived from the measurements strongly suggests that TNFR55 DD adop ts a similar fold in solution. This prompted a homology modeling of the TNF R DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation ( lpr) mutations. Biophysical techniques were used to analyze the effect of c hanging Leu351 to Ala and Leu351 to Asn on the global structure and its imp act on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.