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Structureâactivity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants
- De Wilde, Gert, MurrayâRust, Judith, Boone, Elke, Olerenshaw, Dionne, McDonald, Neil Q., Ibanez, Carlos, Haegeman, Guy, Wollmer, Axel, Federwisch, Matthias
- European journal of biochemistry 2001 v.268 no.5 pp. 1382-1391
- Escherichia coli, antigens, fluorescence emission spectroscopy, gene activation, gene expression, in vivo studies, models, mutants, mutation, neoplasm cells, nerve growth factor, structure-activity relationships, tumor necrosis factors
- Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the soâcalled death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55âDD was expressed and purified as a thioredoxin fusion protein in Escherichia coli.âCircular dichroism, steadyâstate and timeâresolved fluorescence spectroscopy were used to compare TNFR55âDD with DDs of the Fas antigen (Fas), the Fasâassociating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3âdimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55âDD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3âD structure using FADD as a template.âIn vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55âDD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.