Mechanism of Folding and Binding of the N-Terminal SH2 Domain from SHP2.

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.

[1]  C. Pace,et al.  Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding , 1995, Protein science : a publication of the Protein Society.

[2]  L. Mei,et al.  Requirement of SHP2 Binding to Grb2-associated Binder-1 for Mitogen-activated Protein Kinase Activation in Response to Lysophosphatidic Acid and Epidermal Growth Factor* , 2000, The Journal of Biological Chemistry.

[3]  A. Heck,et al.  Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2 , 2005, Chembiochem : a European journal of chemical biology.

[4]  S. Pillai,et al.  Sialic acids and autoimmune disease , 2016, Immunological reviews.

[5]  Hongyang Wang,et al.  Dual faces of SH2-containing protein-tyrosine phosphatase Shp2/PTPN11 in tumorigenesis , 2012, Frontiers of Medicine.

[6]  Sheena E. Radford,et al.  Im7 folding mechanism: misfolding on a path to the native state , 2002, Nature Structural Biology.

[7]  Bruce J Mayer,et al.  What Have We Learned from SH2 Domains? , 2017, Methods in molecular biology.

[8]  A. Fersht,et al.  Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. , 1991, Biochemistry.

[9]  P. Jemth,et al.  A conserved folding mechanism for PDZ domains , 2007, FEBS letters.

[10]  C. Walsh,et al.  The phosphopeptide-binding specificity of Src family SH2 domains. , 1994, Chemistry & biology.

[11]  R. Nussinov,et al.  Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH2 Domains. , 2017, Biophysical journal.

[12]  Andreas Matouschek,et al.  Transient folding intermediates characterized by protein engineering , 1990, Nature.

[13]  Sheena E. Radford,et al.  Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate , 2001, Nature Structural Biology.

[14]  D. Baltimore,et al.  Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides , 1993, Nature.

[15]  K. Jeng,et al.  Effects of peptidic antagonists of Grb2-SH2 on human breast cancer cells. , 2010, Protein and peptide letters.

[16]  S. Khorasanizadeh,et al.  Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues , 1996, Nature Structural Biology.

[17]  M. Brunori,et al.  Identification and characterization of protein folding intermediates. , 2007, Biophysical chemistry.

[18]  John S McMurray,et al.  Targeting SH2 domains in breast cancer. , 2014, Future medicinal chemistry.

[19]  S. Radford,et al.  Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9. , 1999, Journal of molecular biology.

[20]  P. Jemth,et al.  Ligand binding by PDZ domains , 2012, BioFactors.

[21]  S. Marqusee,et al.  Native state energetics of the Src SH2 domain: Evidence for a partially structured state in the denatured ensemble , 2006, Protein science : a publication of the Protein Society.

[22]  M. J. Parker,et al.  An integrated kinetic analysis of intermediates and transition states in protein folding reactions. , 1995, Journal of molecular biology.