Src homology-2 (SH2) domains are small modular domains that recognize phosphotyrosine (pY)-containing proteins and promote the formation of protein complexes, thus playing an essential role in the regulation of many signaling pathways. In this work, the binding specificities of eleven SH2 domains were determined with high resolution using a combinatorial peptide library method. Our results showed subtle yet clear different consensus among SH2 domains. They showed a general preference for a hydrophobic residue at pY+3 position and diverse preferences at pY+1 position. The observed differences were confirmed by surface plasmon resonance (SPR) experiements.
SH2 domain-containing inositol 5-phosphatases 1 (SHIP1) and 2 (SHIP2) have high sequence identity (51%) but share little overlap in their in vivo functions. The sequence specificity of the SHIP2 SH2 domain was defined through the screening of a combinatorial pY peptide library. Comparison of its specificity profile with that of the SHIP1 SH2 domain showed that the two SH2 domains have similar specificities, although there are subtle differences at the pY+1 position. SPR experiments with various pY peptides revealed that the two domains have similar binding affinities but dramatically different binding kinetics, with the SHIP1 SH2 domain having fast association and dissociation rates while the SHIP2 domain showing apparent slow-binding behavior. Mutation of Pro-88 or Pro-105 in the SHIP2 SH2 domain to the corresponding residues in SHIP1 SH2 increased the apparent rate of association and the binding affinity, indicating that cis-trans isomerization of the peptidyl-prolyl bonds is responsible for the observed slow-binding behavior and a potential mechanism for regulating the interaction between SHIP2 and pY proteins. These data suggest that a combination of tissue distribution, specificity, and kinetic differences is likely responsible for their in vivo functional differences.
Grb2 is an adaptor protein that interacts with activated growth factor receptors via its SH2 domain and involved in the mitogenically important Ras signaling pathways. Thus it is an attractive target for the design of inhibitors as anticancer agents. Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. A novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes were synthesized and screened against Grb2 and Tensin SH2 domains. Potent, selective inhibitors were identified from the libraries and were generally more effective than the corresponding linear peptides. One of the inhibitors selected against the Grb2 SH2 domain showed great potency in the inhibition of human breast cancer cell growth and the disruption of actin filaments in vivo.
The structural basis for the ligand-SHP2 NSH2 interaction was investigated by x-ray crystallography. It was found, surprisingly, that the class IV peptide pYFVP bound to SHP2 NSH2 domain at a 2:1 ratio. This unique binding stoichiometry was validated in solution phase by 15N-1H HSQC and 31P NMR experiments. Furthermore, a peptide (pYFLP) derived from TEX10 protein was also proven to have this unique binding stoichiometry.