Abstract
Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors.
Highlights
Protein tyrosine phosphorylation provides a central control mechanism in regulating protein-protein interactions and activation of enzymes in mitogenic signal transduction following activation of cytokine and growth factor receptors [1, 2]
The heat absorbance upon addition of the phosphopeptide to the protein solution underwent a sharp change at 1:1 molar ratio of the protein to peptide, suggesting that the Shc phosphotyrosine binding (PTB) domain binding to the TrkA peptide is very tight, and the stoichiometry of this interaction is 1:1
In this study we have characterized the thermodynamics of binding of the PTB domains of Shc and IRS-1 to the NPXpY motif-containing phosphopeptides
Summary
Protein Preparation—The PTB domain of Shc (residues 17–207) was cloned, expressed, and purified using procedures as described previously [17, 25]. All experiments were carried out at 25 °C in a 50 mM Tris-HCl buffer of pH 8.0 containing 200 mM NaCl, 5 mM -mercaptoethanol, and 1 mM EDTA This condition was optimal for protein stability of the PTB domains of Shc and IRS-1, as there was no sign of significant protein aggregation for up to 0.5–1 mM protein concentration as determined by NMR spectroscopy. Titration curves were fit to an in-built function by a non-linear least squares method using the ORIGIN software (Microcal, Northampton, MA) This function is based upon the binding of a ligand to a macromolecule [26] and contains n (reaction stoichiometry), KD (dissociation constant), and ⌬H (reaction enthalpy) as the variable parameters. The NMR spectra were processed and analyzed using the NMRPipe [27] and NMRView [28] programs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
