Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high a ffinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the c yclic heptapeptide cyclo(CY*VNVPC) by D-alpha-acetylthialysine or D-alpha-l ysine gave cyclo(Y*VNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(Y*VNVP(D-alph a-acetyl-K)) (30), which showed improved binding 10-fold relative to that o f the control peptide KPFY*VNVEF (1). NMR spectroscopy and molecular modeli ng experiments indicate that a beta-turn conformation centered around Y*VNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mo de with a beta-turn conformation. Our data confirm the unique structural re quirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one medi ated by an H2O molecule). These stable and easily accessible cyclic peptide s can serve as templates for the evaluation of phosphotyrosine surrogates a nd further chemical elaboration.