The aim of this proposal is to study the structural, thermodynamic, and kinetic properties of binding of Src Homology 2 (SH2) domains in two protein tyrosine kinase (PTK) systems: Syk and Src. SH2 domains are protein domains which recognize and bind phosphorylated tyrosine residues in specific sequence contexts, thereby allowing protein recruitment onto tyrosine-phosphorylated sties of signaling proteins. Sequences C-terminal to the phosphotyrosine are essential for specific recognition of phosphopeptide targets by SH2 domains. Syk and Src family PTKs cooperate in immune cells to mediate signaling by B- and T-cell receptors: Src family kinases which contain only a single SH2 domain response to receptor activation by phosphorylating tandem repeats of tyrosine residues, which in turn serve as SH2 domain-docking sites for the two SH2 domains of the Syk kinase. The goals of our work are three-fold: 1) establish the energetic principles of peptide binding specificity in SH2 domains using the Src SH2 domain as a model, 2) examine the cooperative interactions between SH2 domains when more than one SH2 domain is present using the tandem- SH2 domain of Syk as a model, and 3) in the longer term, examine the function of SH2 domains in the context of the full-length kinase. The study planned in this proposal aims at dissecting the determinants of macromolecular recognition at tyrosyl-phosphotyrosine by SH2 domains, using an array of methodologies that include the investigation of the role of solutions (ions, pH) and temperature, site-directed mutagenesis of both peptides and proteins, and x-ray crystallography.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060231-03
Application #
6498700
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
2000-02-01
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
3
Fiscal Year
2002
Total Cost
$208,758
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Lubman, Olga Y; Kopan, Raphael; Waksman, Gabriel et al. (2005) The crystal structure of a partial mouse Notch-1 ankyrin domain: repeats 4 through 7 preserve an ankyrin fold. Protein Sci 14:1274-81
Waksman, Gabriel; Kumaran, Sangaralingam; Lubman, Olga (2004) SH2 domains: role, structure and implications for molecular medicine. Expert Rev Mol Med 6:1-18
Kumaran, Sangaralingam; Grucza, Richard A; Waksman, Gabriel (2003) The tandem Src homology 2 domain of the Syk kinase: a molecular device that adapts to interphosphotyrosine distances. Proc Natl Acad Sci U S A 100:14828-33
Lubman, Olga Y; Waksman, Gabriel (2003) Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor. J Mol Biol 328:655-68
Sauer, Frederic G; Pinkner, Jerome S; Waksman, Gabriel et al. (2002) Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation. Cell 111:543-51
Davidson, James P; Lubman, Olga; Rose, Thierry et al. (2002) Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding. J Am Chem Soc 124:205-15
Bradshaw, J Michael; Waksman, Gabriel (2002) Molecular recognition by SH2 domains. Adv Protein Chem 61:161-210
Lubman, Olga Y; Waksman, Gabriel (2002) Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface. J Mol Biol 316:291-304
Grucza, R A; Bradshaw, J M; Mitaxov, V et al. (2000) Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase. Biochemistry 39:10072-81
Bradshaw, J M; Mitaxov, V; Waksman, G (2000) Mutational investigation of the specificity determining region of the Src SH2 domain. J Mol Biol 299:521-35