The beta D-sheet residues of the Lck-derived SH2 domain determine specificity of the interaction with tyrosine-phosphorylated ligands in Ramos B cells.
Src homology 2 (SH2) domains are noncatalytic regions that are conserved among a group of cellular signaling proteins. SH2 domains share the common property of binding phosphotyrosine-containing peptides. Previously, we showed that SH2 domains expressed as recombinant glutathione S-transferase-fusion proteins (GST-SH2) from GTPase-activating protein, Shc, zeta-chain-associated protein tyrosine kinase Zap-70, and Src-like tyrosine kinases precipitated distinct sets of phospho-proteins from activated B cells. To determine the intrinsic structural motifs responsible for the binding specificity within the different SH2 domains, we created chimeric SH2 domains especially focusing on crystal structure-defined contact residues. Recombinant SH2 domains of Lck, Zap-70, and Shc were tested in Ramos B cell lysates for phosphotyrosine-dependent protein binding. Biomolecular interaction analysis (BIAcore) was used to characterize the interaction between the various recombinant SH2 molecules and defined phosphorylated peptides. In agreement with the crystal structure data from the Src and the Lck SH2 domains, our results show that most of the "specificity information" of the Lck SH2 domain is provided by the beta D-sheet, located downstream of the SH2 conserved consensus motif GTFLVRES. In addition, the overall affinity is critically influenced by residues located at the N terminus of the SH2 domain.
von Bonin
A
A
Sandoz Pharma, Ltd., Basel, Switzerland.
Wienands
J
J
Manning
U
U
Zuber
J F
JF
Baumann
G
G
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D016392:Proto-Oncogene Proteins pp60(c-src)