Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display.
We report here on the identification of phosphopetide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand Shc. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.
Gram
H
H
Novartis Pharma AG, Basel, Switzerland.
Schmitz
R
R
Zuber
J F
JF
Baumann
G
G
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D001667:Binding, Competitive; D015374:Biosensing Techniques; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010748:Phosphopeptides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008024:Ligands; D019151:Peptide Library; D010748:Phosphopeptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase