Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase

Abstract

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11 , plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position −2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue −2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues −1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β sheet and the loop that closes the pY binding pocket.

An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

“…Overall, these findings demonstrate that, during the whole simulation, the peptides are tightly bound to the N-SH2 domain. A detailed analysis of ligand–N-SH2 interactions during these simulations are presented elsewhere 30 .…”

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein–Protein Interactions

“… 8 , 10 , 48 , 53 , 54 The crystallographic structures of some of these complexes 8 , 10 , 24 show that an aromatic side chain can be accommodated by a relatively large hydrophobic pocket and that the peptide residue 5 interacts with the BG and EF loops of the domain, which are important for binding specificity. 18 , 24 Finally, a preference for aromatic residues at position +5 has been indicated by several peptide library studies. 36 , 55 − 57 …”

“… 18 , 47 On the basis of our study of the structural determinants of a high affinity for this domain, the IRS-1 pY1172 sequence is near to optimal in several respects, because it has apolar residues at positions +1, +3, and +5, which point toward the hydrophobic groove in the N-SH2 structure, and anionic amino acids at positions +2 and +4, which can interact with a peculiar KxK motif in the BG loop. 18 …”

“…Our previous study clearly indicated that residues −2 to +5 are the most important for the interaction. 18 To clarify the role of N-terminal residues in determining the N-SH2 domain binding affinity, we performed displacement studies ( Figure 2 B) with the unlabeled peptide P9 and with the shortened analogues P8 and P7 ( Table 1 ), where residues −3 or −2 and −3 were removed, respectively. No significant loss of affinity was observed by reducing the sequence to eight residues, while removal of the amino acid at position −2 caused a drastic perturbation of the complex stability ( Figure 2 B).…”

“…However, the N-SH2 domain of SHP2 has a peculiar KxK motif in the region of the BG loop pointing toward the binding groove, which can interact electrostatically with acidic residues present in the peptide sequence at positions +2 and +4. 18 Therefore, by contrast to the superbinders, the high binding affinity of the N-SH2 domain is a result of additional interactions in the selectivity-determining region, and not in the pY pocket. Indeed, our data showed that the pY phosphate contributed <30% of the standard binding free energy.…”