Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2- SH2 domain

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys- Glu(1)- Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr(9)-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 muM) With natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve sidechains of amino acids Glu(1), Tyr(3), Glu(4) Asn(5), and Met(8). Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a P-turn-like conformation. Replacement of Tyr(3) or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu(1), i.e. the amino acid N-terminal to Y-3, remarkably reduces the binding affinity. The position of the Glu(1) side-chain is confirmed to provide a complementary role for pTyr(3), as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 muM) of the nonphosphorylated peptide 11, G1(Gla(1)), in which Glu(1) was replaced by gamma -carboxy- glutamic acid (Gla).

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