Investigation of SH2 domains: Ligand binding, structure and inhibitor design

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 using a combinatorial peptide library method. Three different pY-containing peptide libraries were designed and synthesized to systematically define the binding specificities of SH2 domain. The screening results showed subtle yet clear different consensus among SH2 domains. As revealed from this study, the SH2 domains did not display strong selectivity at positions N-terminal to pY, but they showed much stronger preferences for a hydrophobic residue at pY+3 position. The largest difference was at pY+1 position, where different SH2 domains had different preferences for small residues, positive or negative charged residues, or hydrophilic or hydrophobic residues. The observed subtle differences were confirmed by surface plasmon resonance study.;SH2 domain-containing inositol 5-phosphatases 1 (SHIP1) and 2 (SHIP2) are structurally similar proteins that catalyze the degradation of lipid secondary messenger phosphatidylinositol-3,4,5-triphosphate to produce phosphatidylinositol-3,4-diphosphate. Despite their high sequence identity (51%), SHIP1 and SHIP2 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, both recognizing pY peptides of the consensus sequence pY[S/Y][L/Y/M][L/M/I/V], although there are also subtle differences such as the tolerance of an arginine at the pY+1 position by the SHIP2 but not SHIP1 SH2 domain. Surface plasmon resonance analysis of their interaction 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 (Ser and Glu, respectively) 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 (theoretical diversity of 3.2 x 106) 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 binding specificity of SH2 containing phosphatase 2 (SHP2) N-terminal SH2 was previously determined to have four classes of binding motifs. Peptides from class III and class IV binding motifs were synthesized and crystallized with SHP2 NSH2 domain. It was found, surprisingly, that the class IV peptide pYFVP bound to SHP2 NSH2 domain at 2:1 ratio. This unique binding stoichiometry was validated in solution phase by 15N-1H HSQC and 31P NMR experiments. A peptide (pYFLP) derived from TEX10 was also proven to have this unique binding stoichiometry.