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Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
Published on: September 7, 2011
Thermodynamic characterization of the interaction between TRAF2 and tumor necrosis factor receptor peptides by
1Department of Biochemistry, Weill Medical College of Cornell University, E-023, 1300 York Avenue, New York, NY 10021, USA.
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 microM 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 (DeltaCp = -245 cal/mol small middle dotK). 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.
Insights
Tumor necrosis factor receptor (TNFR) superfamily signaling involves TNF receptor associated factors (TRAFs). This study shows TRAF2 binds weakly to monomeric TNFR peptides, highlighting the importance of receptor clustering for signal transduction.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- The tumor necrosis factor receptor (TNFR) superfamily regulates critical cellular processes like apoptosis and proliferation.
- TNF receptor associated factors (TRAFs) are key signal transducers in TNFR pathways.
- TRAF2's trimeric structure suggests multivalent receptor interactions are crucial for signaling.
Purpose of the Study:
- To thermodynamically characterize the interaction between TRAF2 and monomeric peptide sequences of various TNFR members.
- To investigate the biophysical basis of TRAF2-TNFR interactions and their implications for signal transduction.
Main Methods:
- Isothermal titration calorimetry (ITC) was used to measure binding thermodynamics.
- Peptide sequences from TNFR2, CD40, CD30, Ox40, 4-1BB, and LMP1 were analyzed.
- Thermodynamic data was compared with structural energetics calculations.
Main Results:
- TRAF2 exhibited weak dissociation constants (40 microM to 1.9 mM) with monomeric TNFR peptides.
- Interactions were enthalpy-driven, indicating significant polar contacts.
- A significant temperature dependence of enthalpy (DeltaCp = -245 cal/mol·K) and unfavorable entropy suggest conformational changes.
Conclusions:
- The low affinity of TRAF2 to monomeric peptides underscores the necessity of receptor oligomerization for robust TRAF2 binding and signaling.
- Ligand-induced receptor trimerization or higher-order oligomerization is critical for effective TRAF2 recruitment and signal initiation.
