Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

摘要: Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties SH2 domain protein kinase, we used immobilized phosphopeptides to purified glutathione S-transferase-Src fusion proteins. With this assay, as well free-peptide competition have estimated affinities for various relative SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; = 4 nM). Two Src-derived phosphopeptides, one containing regulatory C-terminal Tyr-527 another autophosphorylation site Tyr-416, specific though low-affinity manner (with about 10(4)-lower affinity than YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide Tyr-857 does not appreciably domain, suggesting it is PDGF-R reported. However, PDGF-R-derived Tyr-751 an approximately orders magnitude lower that YEEI-P). All which contain glutamic acid at position -3 or -4 with respect phosphotyrosine; changing residue alanine greatly diminishes binding. We also tested mutants their interpreted our results light recent crystal structure solution domain. Mutations conserved nonconserved residues (R155A, R155K, N198E, H201R, H201L) cause slight reductions binding, while two mutations severe reductions. The W148E mutant alters invariant tryptophan marks N-terminal border binds poorly phosphopeptides. Inclusion SH3 partially restores by mutant. change arginine coordinates twice phosphotyrosine (R175L) nearly complete loss R175L display high Tyr-751, via least partly independent. show mutation disrupts intramolecular between phosphorylated C terminus within context entire protein; thus, observed vitro assay appear mimic those occur vivo.