Abstract
Src homology 2 (SH2)‐domain‐mediated interactions with phosphotyrosine (pY)‐containing ligands are critical for the regulation of SHP‐1 phosphatase activity. Peptides based on a binding site from receptor tyrosine kinase Ros (EGLN‐pY2267‐MVL, 1) have recently been shown to bind to the SHP‐1 N‐terminal SH2 domain (N‐SH2) with considerably high affinity. In addition, two peptides cyclized between positions −1 and +2 relative to pY (EGLc[K(COCH~2~NH)pYMX]L‐NH~2~, 2: X=D, 3: X=E) bound to the N‐SH2 domain, but did not activate the enzyme and even partially prevented stimulation of SHP‐1 activity by the physiological ligand 1. These findings prompted us to further examine the determinants for optimal binding to the N‐SH2 domain and for the stimulation and inhibition of SHP‐1 activity. Herein we demonstrate that combining the preferred residues in both pY+1 (such as Phe or norleucine, Nle) and pY+3 (such as homophenylalanine, Hfe) leads to highly efficient activating ligands of SHP‐1. Particularly in the context of the cyclic peptides 7 (EGLc[K(COCH~2~NH)pYFD]Hfe‐NH~2~) and 8 (EGLc[K(COCH~2~NH)pYNleD]HfeL‐NH~2~), the incorporation of these residues resulted in high‐affinity ligands with a significantly increased ability to stimulate SHP‐1 activity. We suggest that different binding modes (according to consensus sequences class I and II) are responsible for obtaining either activating (7 and 8) or nonactivating (2 and 3) ligands. Peptides such as 7 and 8 that bind in the extended fashion of the type II mode activate the phosphatase through complete filling of the cavity for pY+3. In contrast, peptides such as 2 and 3 that bind in the class I mode do not activate the enzyme because they allow more conformational space at pY+3. Therefore, their binding does not force the conformational transition necessary to trigger the dissociation of N‐SH2 and the catalytic domain.