Gain-Of-Function SHP2 E76Q Mutant Rescuing Autoinhibition
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Article Cite This: J. Chem. Inf. Model. 2019, 59, 3229−3239 pubs.acs.org/jcim Gain-of-Function SHP2 E76Q Mutant Rescuing Autoinhibition Mechanism Associated with Juvenile Myelomonocytic Leukemia † ‡ ‡ † † † † Ashfaq Ur Rehman, , Humaira Rafiq, Mueed Ur Rahman, Jiayi Li, Hao Liu, Shenggan Luo, † ‡ † Taaha Arshad, Abdul Wadood, and Hai-Feng Chen*, † State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China ‡ Department of Biochemistry, Abdul Wali Khan University, Mardan 23200, Pakistan *S Supporting Information ABSTRACT: Juvenile myelomonocytic leukemia (JMML) is an invasive myeloproliferative neoplasm and is a childhood disease with very high clinical lethality. The SHP2 is encoded by the PTPN11 gene, which is a nonreceptor (pY)- phosphatase and mutation causes JMML. The structural hierarchy of SHP2 includes protein tyrosine phosphatase domain (PTP) and Src-homology 2 domain (N-SH2 and C- SH2). Somatic mutation (E76Q) in the interface of SH2-PTP domain is the most commonly identified mutation found in up to 35% of patients with JMML. The mechanism of this mutant associated with JMML is poorly understood. Here, molecular dynamics simulation was performed on wild-type and mutant (E76Q) of SHP2 to explore the precise impact of gain-of-function on PTP’s activity. Consequently, such impact rescues the SHP2 protein from autoinhibition state through losing the interface interactions of Q256/F7 and S502/Q76 or weakening interactions of Q256/R4, Q510/G60, and Q506/A72 between N-SH2 and PTP domains. The consequences of these interactions further relieve the D′E loop away from the PTP catalytic site. The following study would provide a mechanistic insight for better understanding of how individual SHP2 mutations alter the PTP’s activity at the atomic level. ■ INTRODUCTION However, the C-SH2 domain does not play a direct role in 4 The SHP2 is a human cytoplasmic protein tyrosine the catalytic activity of SHP2. However, its indirect role is phosphatase, encoded by PTPN11 gene. The structural binding of the bisphosphoryl ligand to one site; the C-SH2 components/constituents/composition/order of SHP2 in- domain provides binding energy and increases the local concentration of ligand, so that the compromised N-SH2 Downloaded via SHANGHAI JIAO TONG UNIV on December 6, 2019 at 05:34:42 (UTC). cludes a catalytic domain (PTP) and a regulatory domain 4 (SH2)1 that were positioned at amino and carboxyl terminal of domain can bind a second site. Upon binding to the second See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 2,3 site, the N-SH2 domain adopts the A-state (activated); this SHP2 protein, respectively. In the basal state, the N-SH2 4 domain packs against the catalytic cleft of PTP domain, and shifts the equilibrium to the open, active form of the PTP. sterically inhibits the catalytic activity by inserting a D′E loop Engagement of the C-SH2 domain alone is unlikely to 4 contribute to activation because it makes only minimal contact (Residues N58, T59, G60, D61, Y62, and A72) into the 4 catalytic cleft (Residues C459, D464, R465, Q506, and with the other domains. SHP2 is a signal-enhancing G503),4 which subsequently reduces the solvent accessibility component (growth factor, cytokine, and extra-cellular matrix and thus blocks access of substrate to the catalytic site.4 receptor). Signaling expressed ubiquitously and plays an essential role in regulating cell proliferation, differentiation, Engagement of SHP2 SH2 domains with phosphotyrosine 1 ligands has been observed to increase phosphatase activity,5,6 and migration. The SHP2 can also regulate several down- and previous research has revealed that many leukemia- stream signaling cascades such as RAS-ERK, PI3K-AKT, and JAK-STAT; however, its role in these pathways is as a receptor associated SHP2 mutations enhance sensitivity to phospholi- 1,8 gand activation relative to that of the wild-type enzyme.7 This and/or cell context-dependent. Overactivation of SHP2 due binding event or induced point mutation disrupts the interface to germline mutation in PTPN11 has been observed in some interaction among N-SH2 and PTP and further activate the abnormal states like, 35% in hematological malignancies phosphatase. Thus, the N-SH2 domain is a conformational switch that binds and inhibits phosphatase or binds Received: April 25, 2019 phosphotyrosyl peptide (pY) and activates the enzymes. Published: June 20, 2019 © 2019 American Chemical Society 3229 DOI: 10.1021/acs.jcim.9b00353 J. Chem. Inf. Model. 2019, 59, 3229−3239 Journal of Chemical Information and Modeling Article counting juvenile myelomonocytic leukemia, 7% in B-cell acute package.22 The LEaP module was used to add hydrogen atoms lymphoblastic leukemia, 50% in the developmental Noonan to both crystallographic structures of SHP2 protein. Counter- syndrome disorder,9 10% in myelodysplastic syndrome, low ions (Na+ and Cl−) were added to maintain system neutrality. occurrence in solid tumors, and less than 5% in acute myeloid All systems were solvated in a truncated octahedral box of leukemia.10,11 TIP3P water model with 10 Å buffer. Particle mesh Ewald Earlier literature has identified that many mutations (D61G, (PME)23 method was used to treat long-range electrostatic − D61Y, and E76K) are associated with cancerous/carcino- interactions and ff 99IDPs force field24 26 was used for all genic.12 Thus, PTPN11 is recognized as the first proto- simulations. All the bonds involving hydrogen atoms were oncogene.13 However, the activation mechanism for the constrained with SHAKE algorithm.27 following mutations has been explored using MD simulation PMEMD of CUDA version was used to accelerate all the − by Wang et al.14 16 In the present study, we have explored the MD simulations.28 Steepest descent method was used to activation mechanism for the first time, induced by GOF- minimize the solvated systems for 20 000-step, then 400 ps mutation (E76Q) in SHP2 protein using essential dynamics heating, and 200 ps equilibration in the NVT ensemble. The simulation approaches. This mutation has been identified in defaults parameters of the Langevin thermostat method for exon 3 with some additional mutations that encrypt the N-SH2 heating and equilibration runs with a time step of 2 fs was regulatory domain. Highly prophetic of GOF, the consequen- used, and for the production runs under NPT ensemble at 298 ces of this mutation at position 76 are hence to activate the K with a time step of 2 fs in Berendsen’s thermostat and SHP2 protein productively. barostat. Additionally, as a consequence of this mutation, the interface Essential Dynamics Analysis of the MD Trajectories. residue−residue interactions were diminished, which further Dominant motions in both SHP2+E76 and SHP2+Q76 systems increased the solvent accessibility of the catalytic site, were analyzed through essential dynamics analysis for the providing a definite clue about SHP2 protein active state.4 trajectory file.29,30 Then the positional covariance matrix of Previous works have demonstrated that E76Q mutant of SHP2 atomic coordinates was calculated including their eigenmodes. is found within 91% acute myeloid leukemia samples with 6.6% Subsequently, the extracted data for the matrix was mutation rate.17 Based on the saturation analysis of cancer diagonalized by an orthogonal coordinate transformation genes in AML, these PTPN11 mutations are predicted to be yielding the diagonal matrix of eigenvector and its values. functionally important. A significant shift of equilibrium The first eigenmode and its value generally specify the favoring active conformation in SHP2+Q76 caused GOF. principal component of the trajectory, which contains the Although it has been reported that SHP2+Q76 protein activity substantial dominant motion of structures. The extent and has been enhanced due to the CA distance between the direction of the most dominant motions of all systems were interface residues (N-SH2 and PTP) that tremendously visualized through porcupine plots using the ‘modevectors.py’ increased. The consequent differences in CA distance among script (written by Sean M. Law) in PyMol v1.7.31 PTP and N-SH2 domain are that some paired residues lose Free energy (FE) landscape was plotted with the first two (Q256/F7 and S502/Q76) or weakened (Q256/R4, Q510/ principal components (PC1 and PC2). The corresponding G60, and Q506/A72) the hydrogen interaction which Gibbs energy represents conformations of molecules obtained ultimately open up the catalytic site for the substrate. The through the trajectory. The deep valleys represent stable and dynamic mechanical behavior of polymorphic E76Q mutation dominant conformations, and boundaries represent intermedi- is still unclear. Therefore, the molecular dynamic (MD) ate conformations of the molecules.32 The ‘g_sham’ function simulations of the mutant (SHP2+Q76)andwild-type distributed in GROMACS was used for the Gibbs energy (SHP2+E76) system were used to reveal the entire physiological calculations, and Origin v9.1 was used to obtain 3D images of mechanism of SHP2 protein. the plots. Coordinates on 3D images were used to find exact Molecular dynamic (MD) simulations have widely been time frames and snapshots of molecules at a time and state. used to study the structural differences at the atomic level and Dynamic Cross-Correlation Map. To better understand were successfully used to gain insight into the impact of a point the dynamics of the SHP2+E76 structure and SHP2+Q76 mutation at the interface between N-SH2 and PTP structure, DCCM were generated to analyze the cross- domain.18,19 The consequent impact further enhanced the correlation shift of the backbone atoms (Cα), that can provide solvent accessibility of the catalytic cleft region in the PTP information about the effect of mutations on protein dynamics domain, and hence, both the domains moved away from each by analyzing how atomic displacement was coupled.33 The other to rescue the SHP2 protein from autoinhibition state.