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Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central β-sheet.

ABSTRACT: SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central β-sheet, and that partial unzipping of this β-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

SUBMITTER: Marasco M

PROVIDER: S-EPMC10951427 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature

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Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central β-sheet.

Marasco Michelangelo M Kirkpatrick John J Carlomagno Teresa T Hub Jochen S JS Anselmi Massimiliano M

Computational and structural biotechnology journal 20240302

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crys ...[more]

PMID: 38510972

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Project description: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.

Project description:BackgroundWe have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence.ObjectiveTo determine the effect of SHP2 on fibrotic responses.Methods and resultsPrimary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy.ConclusionsSHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases.

Dataset Information

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central β-sheet.

Publications

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central β-sheet.

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