Alternative Mode of Binding to Phosphotyrosyl Peptides by Src Homology-2 Domains

Abstract

Src homology-2 (SH2) domains recognize specific phosphotyrosyl (pY) proteins and promote protein-protein interactions. In their classical binding mode, the SH2 domain makes specific contacts with the pY residue and the three residues immediately C-terminal to the pY, although for a few SH2 domains, residues N-terminal to pY have recently been shown to also contribute to the overall binding affinity and specificity. In this work, the ability of an SH2 domain to bind to the N-terminal side of pY has been systematically examined. A pY peptide library containing completely randomized residues at positions -5 to -1 (relative to pY, which is position 0) was synthesized on TentaGel resin and screened against the four SH2 domains of phosphatases SHP-1 and SHP-2. Positive beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay, and the peptides were sequenced by partial Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The N-terminal SH2 domain of SHP-2 binds specifically to peptides of the consensus sequence (H/F)XVX(T/S/A)pY. Further binding studies with individually synthesized pY peptides show that pY and the five residues N-terminal to pY, but not any of the C-terminal residues, are important for binding. The other three SH2 domains also bound to the library beads, albeit more weakly, and the selected peptides did not show any clear consensus. These results demonstrate that at least some SH2 domains can bind to pY peptides in an alternative mode by recognizing only the residues N-terminal to pY.

Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes

“…3B). Similar differences were consistently observed for the total variance of the coordinates of the N-SH2 C a atoms (SHP2 I42 : 157 Å 2 ; wild-type SHP2: 119 Å 2 ; SHP2 A42 : 85 Å 2 ), implying that the higher peptide mobility was correlated to a higher level of protein fluctuations, particularly in the BC loop (residues 34-40), N-terminal helix (residues [13][14][15][16][17][18][19][20][21][22] and the loop preceding it (residues 9-12) (Fig. 3B).…”

Sorafenib analogue SC‐60 induces apoptosis through the SHP‐1/STAT3 pathway and enhances docetaxel cytotoxicity in triple‐negative breast cancer cells

“…SHP-1 is composed of two SH-2 domains (N-SH2 and C-SH2) and one catalytic PTP domain (Yang et al, 1998). Studies have shown that the activity of SHP-1 is also regulated by the conformational rearrangement upon substrate/chemical binding: The N-SH2 domain is released from the interaction with the PTP domain exposing the catalytic site of the PTP domain, thereby enhancing SHP-1 activity (Qin et al, 2005;Wang et al, 2011;Yang et al, 2003). Interestingly, we identified that multiangiokinase inhibitor sorafenib also acts as a direct enhancer of SHP-1 (Tai et al, 2011(Tai et al, , 2014b.…”

Disrupting VEGF-A paracrine and autocrine loops by targeting SHP-1 suppresses triple negative breast cancer metastasis

“…Secondly, the regulatory network of SHP-1 with other molecules (tumor suppressors or oncoproteins) that might influence patient prognosis remains to be defined. Moreover, biologically the activity of SHP-1 strongly depends on its structure conformation change (open and close forms), such as ligand and chemicals binding and thus expression levels not necessarily correlated with SHP-1 activities 24 25 26 27 ; however, it is difficult to directly measure SHP-1 activity from archived patient tumor samples. Nevertheless, more studies are necessary to determine the significance of SHP-1 expression in correlation with survival outcome in patients with TNBC.…”

“…The activity of SHP-1 strongly depends on the conformational rearrangement through ligand or chemical binding. The interaction between ligands/chemicals and SH2 domains will induce conformational changes of SHP-1, resulting in the releasing of the N-SH2 domain from the PTP domain and initiating the whirling of the C-SH2 domain and further exposing the active site of PTP domain 25 26 27 . Interestingly, we previously reported that regorafenib could directly activate SHP-1 in HCC and CRC, which was independent of its angio-kinase inhibition 28 29 .…”