Abstract
The SH2 domain of Grb2 binds phophotyrosyl peptides with the consensus sequence pYXNX within several proteins including the adapter proteins Shc, FRS-2, growth factor receptors such as the members of the erbB family, morphology-determining proteins such as FAK, and cellular oncogenes such as Bcr-Abl. Binding of the Grb2 SH2 domain to the receptors relocates the Grb2 SH3 domain binding proteins, i.e. Sos, close to the plasma membrane. Then, Sos due to its guanine nucleotide exchange activity, converts the GDP-bound inactive form of Ras to its GTP-bound active form. Activated Ras triggers the kinase cascade which is essential for cell growth and differentiation. A particularly important role for Grb2 in human cancer has been proposed for cells transformed by high levels of erbB2 (HER-2 or neu) expression. Recent studies have indicated that Grb2 function is required for cell transformation by the neu and Bcr-Abl oncogenes. Thus, the design of specific inhibitors to Grb2 SH2 domain holds the promise of targeted treatment of breast cancer and cancer. Based on circular dichroism measurements, its melting temperature was found to be 55℃, at pH7.4. The Gibbs free energy (△GH2O) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 1.19 kcal/mole by Gdn×HCl denaturation experiments, as compared to 3.96 kcal/mole (BTK SH3 domain) and 3.03 kcal/mole (Itk SH3 domain)[1], indicating poor stability of Grb2 SH2 domain. Until now, most of the inhibitors designed for the SH2 domain of Grb2 are focused on the phosphotyrosine containing peptides and peptidomimetics. However, these compounds all face the stability (chemical and/or biological) of the pTyr side chain phosphate ester and the poor cellular penetrability problems. Recently, nonphosphorylated cyclic peptide ligands were discovered for the Grb2 SH2 domain using phage display library. G1TE, One of these cyclic peptides, was determined using two-dimensional NMR and simulated annealing methods. The average RMS-deviations were found to be 0.41±0.11 A for the backbone heavy atoms C, Cα and N, and 1.03±0.14 A for all heavy atoms in a family of 10 structures. Structural comparison between G1TE and Bcr-Abl phosphopeptide bound to Grb2 SH2 domain revealed that G1TE may form a larger circle-like binding surface than the Bcr-Abl phosphopeptide. Since these cyclic peptides do not contain pTyr, they define a new type of SH2 domain binding motif that may advance the design of Grb2 inhibitors.