Alternative modes of binding of proteins with tandem SH2 domains.
The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.
O'Brien
R
R
Department of Biochemistry and Molecular Biology, University College London, United Kingdom.
Rugman
P
P
Renzoni
D
D
Layton
M
M
Handa
R
R
Hilyard
K
K
Waterfield
M D
MD
Driscoll
P C
PC
Ladbury
J E
JE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry*; D013816:Thermodynamics; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase