Open AccessStructure–activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants
Author(s) -
De Wilde Gert,
MurrayRust Judith,
Boone Elke,
Olerenshaw Dionne,
McDonald Neil Q.,
Ibanez Carlos,
Haegeman Guy,
Wollmer Axel,
Federwisch Matthias
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2001.02004.x
Subject(s) - death domain , fadd , receptor , tumor necrosis factor alpha , microbiology and biotechnology , mutant , in vivo , circular dichroism , biology , fusion protein , chemistry , apoptosis , programmed cell death , gene , biochemistry , immunology , recombinant dna , genetics , caspase
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 so‐called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli . Circular dichroism, steady‐state and time‐resolved fluorescence spectroscopy 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, for which the 3‐dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR 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 changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact 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.