Development of Antagonist Peptides of the Crk/CrkL SH2 Domains by Modifying the Peripheral Amino Acid Residues of the YXXP Motif.

Protein-protein interactions (PPIs) through the phosphotyrosine-Src homology 2 (SH2) interaction play critical roles in many signal transduction pathways. CT10 regulator of kinase (Crk) and Crk-like (CrkL) are structurally and functionally similar adaptor proteins that mediate PPIs through their SH2 domains. Crk and CrkL are essential in tumor cell migration and invasion and have been proposed as therapeutic targets. However, there are no Crk and CrkL inhibitors available. Previously, we designed a conserved heptapeptide that binds to Crk by analyzing the 15 YXXP motifs in p130Cas. The peptide bound to the SH2 domain of Crk (CrkSH2) and inhibited the CrkSH2-p130Cas interaction. Here, to design peptides with higher affinities, we performed two rounds of the structure-function relationship study in which the nonconserved amino acid residues were substituted with other l-amino acids based on molecular modeling results. The binding activities of the substituted peptides were determined by saturation transfer difference NMR (STD-NMR), fluorescence polarization (FP), and FP competition assays. Whereas none of the core amino acid substitutes improved the binding affinities, two terminal amino acid substitutes improved the binding affinities to Crk and CrkL and inhibited the Crk/CrkL-mediated PPIs. Our study demonstrates that substitution of amino acids at the -2 and +4 positions from the conserved phosphotyrosine improves the binding affinity of the YXXP motif-containing peptide to the SH2 domains of Crk and CrkL. Our study proposes modification or optimization of the peripheral amino acid residues of the YXXP motif to improve the YXXP-SH2 binding and develop inhibitors of Crk and CrkL.