title: Thermodynamic study of the binding of the tandem-SH2 domain of the Syk kinase to a dually phosphorylated ITAM peptide: evidence for two conformers.
abstract: 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.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Grucza
authors.forename: K
authors.initials: K
authors.lastname: Fütterer
authors.forename: A C
authors.initials: AC
authors.lastname: Chan
authors.forename: G
authors.initials: G
authors.lastname: Waksman
mesh_terms: D017252:CD3 Complex; D002942:Circular Dichroism; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D017459:Receptors, Amino Acid / Q000737:chemistry / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism*; D000072377:Syk Kinase; D013696:Temperature; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D017252:CD3 Complex; C115335:CD3E protein, human; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D017459:Receptors, Amino Acid; D011948:Receptors, Antigen, T-Cell; C087659:tyrosine receptor; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase