Thermodynamic characterization of the interaction between TRAF2 and tumor necrosis factor receptor peptides by isothermal titration calorimetry

Abstract

The tumor necrosis factor receptor (TNFR) superfamily can induce diverse biological effects, including cell survival, proliferation, differentiation, and apoptosis. The major signal transducers for TNFRs are the family of TNF receptor associated factors (TRAFs). The direct interaction between TRAFs and the intracellular tails of TNFRs is the first step of this signal relay process. Structural studies have revealed a trimeric nature of TRAF2 and a symmetrical mode of receptor binding, suggesting the involvement of trivalent TNFR2-receptor interaction in the signal transduction. In this study, using isothermal titration calorimetry (ITC), we report thermodynamic characterization of the interaction between TRAF2 and monomeric peptide sequences from TNFR members, including TNFR2, CD40, CD30, Ox40, and 4-1BB, and the Epstein-Barr virus (EBV)-transforming protein, latent infection membrane protein-1 (LMP1). The dissociation constants of the interaction were shown to range between 40 M and 1.9 mM, which are substantially weaker than most protein-peptide interactions. The interaction is entirely driven by exothermic enthalpy, consistent with the abundance of polar contacts. The enthalpy of the interaction has a significant temperature dependence (⌬Cp ‫؍‬ ؊245 cal͞mol⅐K). The unfavorable entropy in the interaction and the comparison with structural energetics calculations suggest the involvement of conformational rearrangement in the interaction. The low affinity of TRAF2 to monomeric receptor peptides further supports the importance of avidity contribution in TRAF2 recruitment by these receptors upon ligand-induced trimerization or higher order oligomerization. F or a multicellular organism, receptor signal transduction through a membrane barrier is one of the most intriguing aspects of the communication between individual cells. Many elegant studies have revealed that conformational changes and receptor clustering are the two most frequently observed mechanisms of receptor signaling. Neurotransmitters and many peptide ligands bind and induce conformational changes to their transmembrane receptors and activate the G proteins associated with the intracellular domains of these receptors (1). The initiation of signal transduction by many growth factor receptors is by ligand-mediated receptor dimerization, which brings the intracellular tyrosine kinase domains into proximity for autophosphorylation and enzymatic activation (2).The signal transduction of the tumor necrosis factor receptor (TNFR) superfamily, a group of receptors involved in cell survival and cell death (3), is also thought to be induced by ligand-induced receptor clustering (4). The extracellular domains of TNFRs are composed of differing numbers of cysteinerich repeats and share extensive sequence homology (5). The ligands for TNFRs belong to the corresponding TNF superfamily and are trimeric in nature (6). The extracellular interaction of a TNF-like ligand with a TNFR indirectly trimerizes the receptor by imposing a 3-fold symmetry to the interaction. In t...

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts

“…TRAFs are trimeric intermediates in the signaling pathway of numerous TNF receptor family members. 41 By bringing together 3 receptors, a trimeric ligand may allow recruitment to the complex of just one intracellular TRAF, which may not be enough to induce signaling, as oligomerization of trimeric TRAF-2 and TRAF-6 is required to activate downstream signaling events. 42 BAFF-R only interacts with TRAF-3, whose mode of action and multimerization requirements may be distinct, making BAFF-R-mediated signals less dependent on ligand oligomerization.…”

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma

“…Trimerization or multimerization of TNFR/tumor necrosis receptor-associated factors (TRAFs) is required for high-affinity interaction. 16,17 The study of CD40/CD40L, one of the TNFR/TRAFs members, mAb indicated that bivalent mAb and dimeric or trimeric soluble forms of CD40L exhibit more potent biological activity than monomeric soluble CD40L. 18 Furthermore, the Fc region of mouse Ig may increase the stability of 4-1BBL and also serves as a tag for purification via a protein G column.…”

TRAF1 Is a Critical Regulator of JNK Signaling by the TRAF-Binding Domain of the Epstein-Barr Virus-Encoded Latent Infection Membrane Protein 1 but Not CD40

“…Thus, while TRAF1 directly binds CTAR1 (7), it associates indirectly with CD40 and, presumably, with the CTAR2-TRADD complex via TRAF2 (36,50). Furthermore, TRAF2 strongly binds the N terminus of TRADD (46,55) and a PxQxT motif in the CD40 cytoplasmic tail (50) but only weakly interacts with a PxQxxD motif in LMP1 CTAR1 (7,52,63,64). It is therefore possible that TRAF1 may influence the affinity or avidity of TRAF2 for CTAR1.…”