Thermodynamic study of the binding of the tandem-SH2 domain of the Syk kinase to a dually phosphorylated ITAM peptide: evidence for two conformers.

The cytosolic tyrosine kinase Syk is recruited to immune cell receptors via interactions of its tandem-SH2 domain with tyrosine-phosphorylated sequences called immune receptor tyrosine activation motifs (ITAMs). We have characterized the binding of the tandem-SH2 domain of Syk (Syk-tSH2) to a dually phosphorylated peptide derived from the ITAM of the T cell receptor CD3-epsilon subunit. The CD3-epsilon peptide binds with an affinity of 18-81 nM at 150 mM NaCl over the 4.5-30 degrees C temperature range that was studied. The enthalpy of binding, DeltaH degrees obs, shows an unusual nonlinear dependence on temperature, suggesting the possibility of a temperature-dependent conformational equilibrium coupled to binding. This hypothesis was tested and confirmed by examining the temperature dependence of (1) the on-rate constant for binding and (2) the fluorescence of Syk-tSH2 and its CD3-epsilon peptide complex. The DeltaH degrees obs, Kobs, fluorescence, and kinetic data are all well described by a model incorporating the hypothesized conformational equilibrium. Circular dichroism spectra at various temperatures indicate that the conformational change is not due to a partial unfolding of the protein. We suggest that the conformational equilibrium enables Syk-tSH2 to exhibit flexibility in its binding modality, which may be necessitated by Syk's involvement in a wide variety of signal tranduction pathways.