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-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-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 microM) 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 side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. 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 beta-turn-like conformation. Replacement of Tyr3 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 Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).
Lung
F D
FD
Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Frederick, USA.
Long
Y Q
YQ
Roller
P P
PP
King
C R
CR
Varady
J
J
Wu
X W
XW
Wang
S
S
D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D008024:Ligands; D008958:Models, Molecular; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; D008024:Ligands; D019151:Peptide Library; D010456:Peptides, Cyclic; D011506:Proteins