R.A. Grucza, K. Futterer, A.C. Chan, and G. Waksman.
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 or 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 18-81 nM affinity at 150 mM NaCl
over the 4.5 to 30 degree C temperature range studied. The enthalpy of binding, delta-Hobs,
shows an unusual non-linear 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 deltaHobs, Kobs ,
fluorescence, and kinetic data are all well described by a model incorporating the
hypothesized conformational equilibrium. Circular dichroism spectra at various
temperature 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.
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