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

G De Wilde; J Murray-Rust; E Boone; D Olerenshaw; N Q McDonald; C Ibanez; G Haegeman; A Wollmer; M Federwisch

doi:10.1046/j.1432-1327.2001.02004.x

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

Eur J Biochem. 2001 Mar;268(5):1382-91. doi: 10.1046/j.1432-1327.2001.02004.x.

Authors

G De Wilde¹, J Murray-Rust, E Boone, D Olerenshaw, N Q McDonald, C Ibanez, G Haegeman, A Wollmer, M Federwisch

Affiliation

¹ Department of Molecular Biology, University of Gent-VIB, Belgium.

PMID: 11231290
DOI: 10.1046/j.1432-1327.2001.02004.x

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing*
Amino Acid Sequence
Animals
Antigens, CD / chemistry*
Antigens, CD / genetics
Antigens, CD / metabolism*
Carrier Proteins / chemistry
Circular Dichroism
Escherichia coli
Fas-Associated Death Domain Protein
Guanidine / pharmacology
Humans
Mice
Models, Molecular
Molecular Sequence Data
Mutation / genetics*
Phenotype
Protein Denaturation / drug effects
Protein Structure, Secondary
Protein Structure, Tertiary
Receptor, Nerve Growth Factor / chemistry
Receptors, Tumor Necrosis Factor / chemistry*
Receptors, Tumor Necrosis Factor / genetics
Receptors, Tumor Necrosis Factor / metabolism*
Receptors, Tumor Necrosis Factor, Type I
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / metabolism
Sequence Alignment
Signal Transduction*
Spectrometry, Fluorescence
Structure-Activity Relationship
Thioredoxins / chemistry
Thioredoxins / genetics
Thioredoxins / metabolism
Tumor Cells, Cultured
fas Receptor / chemistry

Substances

Adaptor Proteins, Signal Transducing
Antigens, CD
Carrier Proteins
FADD protein, human
Fadd protein, mouse
Fas-Associated Death Domain Protein
Receptor, Nerve Growth Factor
Receptors, Tumor Necrosis Factor
Receptors, Tumor Necrosis Factor, Type I
Recombinant Fusion Proteins
fas Receptor
Thioredoxins
Guanidine