Phosphopeptide ligands of the SHP-1 N-SH2 domain: effects on binding and stimulation of phosphatase activity.
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.
Hampel
Kornelia
K
Institute of Biochemistry and Biophysics, Biological and Pharmaceutical Faculty, Friedrich Schiller University, Philosophenweg 12, 07743 Jena, Germany.
Kaufhold
Ina
I
Zacharias
Martin
M
Böhmer
Frank D
FD
Imhof
Diana
D
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D047908:Intracellular Signaling Peptides and Proteins / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010456:Peptides, Cyclic / Q000737:chemistry / Q000494:pharmacology*; D010748:Phosphopeptides / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics; D018909:src Homology Domains
D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010456:Peptides, Cyclic; D010748:Phosphopeptides; C514099:glutamyl-glycyl-leucyl-cyclo(lysyl(COCH2NH)-phosphotyrosyl-methionyl-aspartyl)-leucinamide; C514100:glutamyl-glycyl-leucyl-cyclo(lysyl(COCH2NH)-phosphotyrosyl-methionyl-glutamtyl)-leucinamide; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases