ABSTRACT
SHP2 is a phosphatase protein, involved in many cellular pathways, comprising two SH2 domains (namely N-SH2 and C-SH2) and a phosphatase domain. Among others, the interaction between SHP2 and Gab2 (Grb2 associated binder) is critical in cell death and differentiation. SHP2 binds to Gab2 through its SH2 domains, which recognize specific regions of Gab2 characterized by the presence of a phosphorylated tyrosine. In order to shed light on the dynamic and functional properties of this protein-protein interaction, we studied the mechanism of folding of N-SH2 and the binding process to a peptide mimicking a region of Gab2. The data presented represent the first description by stopped-flow of the kinetics of binding of an SH2 domain in solution. By performing experiments at different ionic strengths, we elucidate the electrostatic nature of the interaction, highlighting a key role of the negative charge of the phosphotyrosine in the recognition event of the reaction. Furthermore, by analyzing the equilibrium and kinetics of folding of N-SH2 folding we demonstrate the presence of an intermediate along the folding pathway. These results are discussed in the light of previous works on another SH2 domain.
Subject(s)
Protein Folding , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , src Homology Domains , Humans , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 11/isolation & purification , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolismABSTRACT
A method for 2-SH2 protein domain study was described as per the order of expression, purification and structural detection. The 2-SH2 protein of Homo sapiens SHP-2 was successfully expressed and purified. It could specifically bind to anti-SHP-2/SHPTP-2 antibody according to the MS and Western blot analysis. The NMR spectrum result reveals that the protein exists in a well-ordered structure. This can provide foundations to find out the reaction mechanism of the D phosphorylated-EPIYA motif accessible to 2-SH2, support the research and development of the novel detection chip as well as target inhibition medicine for the future clinical applications.