Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

SHP2-EGFR States in Dephosphorylation Can Inform Selective SHP2 Inhibitors, Dampening RasGAP Action.

SHP2 is a positive regulator of the EGFR-dependent Ras/MAPK pathway. It dephosphorylates a regulatory phosphorylation site in EGFR that serves as the binding site to RasGAP (RASA1 or p120RasGAP). RASA1 is activated by binding to the EGFR phosphate group. Active RASA1 deactivates Ras by hydrolyzing Ras-bound GTP to GDP. Thus, SHP2 dephosphorylation of EGFR effectively prevents RASA1-mediated deactivation of Ras, thereby stimulating proliferation. Despite knowledge of this vital regulation in cell life, mechanistic in-depth structural understanding of the involvement of SHP2, EGFR, and RASA1 in the Ras/MAPK pathway has largely remained elusive. Here we elucidate the interactions, the factors influencing EGFR's recruitment of RASA1, and SHP2's recognition of the substrate site in EGFR. We reveal that RASA1 specifically interacts with the DEpY992LIP motif in EGFR featuring a proline residue at the +3 position C-terminal to pY primarily through its nSH2 domain. This interaction is strengthened by the robust attraction of two acidic residues, E991 and D990, of EGFR to two basic residues in the BC-loop near the pY-binding pocket of RASA1's nSH2. In the stable precatalytic state of SHP2 with EGFR (DADEpY992LIPQ), the E-loop of SHP2's active site favors the interaction with the (-2)-position D990 and (-4)-position D988 N-terminal to pY992 in EGFR, while the pY-loop constrains the (+4)-position Q996 C-terminal to pY992. These specific interactions not only provide a structural basis for identifying negative regulatory sites in other RTKs but can inform selective, high-affinity active-site SHP2 inhibitors tailored for SHP2 mutants.

Molecular recognition of ITIM/ITSM domains with SHP2 and their allosteric effect.

Src homology 2-domain-containing tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase that is widely expressed in a variety of cells and regulates the immune response of T cells through the PD-1 pathway. However, the activation mechanism and allosteric effects of SHP2 remain unclear, hindering the development of small molecule inhibitors. For the first time, in this study, the complex structure formed by the intact PD-1 tail and SHP2 was modeled. The molecular recognition and conformational changes of inactive/active SHP2 versus ITIM/ITSM were compared based on prolonged MD simulations. The relative flexibility of the two SH2 domains during MD simulations contributes to the recruitment of ITIM/ITSM and supports the subsequent conformational change of SHP2. The binding free energy calculation shows that inactive SHP2 has a higher affinity for ITIM/ITSM than active SHP2, mainly because the former's N-SH2 refers to the α-state. In addition, a significant decrease in the contribution to the binding energy of certain residues (e.g., R32, S34, K35, T42, and K55) of conformationally transformed SHP2 contributes to the above result. These detailed changes during conformational transition will provide theoretical guidance for the molecular design of subsequent novel anticancer drugs.

Single Ion Pair Is Essential for Stabilizing SHP2's Open Conformation.

Src-homology-2-domain-containing PTP-2 (SHP2) is a widely expressed signaling enzyme whose misregulation is associated with multiple human pathologies. SHP2's enzymatic activity is controlled by a conformational equilibrium between its autoinhibited ("closed") state and its activated ("open") state. Although SHP2's closed state has been extensively characterized, the putative structure of its open form has only been revealed in the context of a highly activated mutant (E76K), and no systematic studies of the biochemical determinants of SHP2's open-state stabilization have been reported. To identify amino-acid interactions that are critical for stabilizing SHP2's active state, we carried out a mutagenic study of residues that lie at potentially important interdomain interfaces of the open conformation. The open/closed equilibria of the mutants were evaluated, and we identified several interactions that contribute to the stabilization of SHP2's open state. In particular, our findings establish that an ion pair between glutamate 249 on SHP2's PTP domain and arginine 111 on an interdomain loop is the key determinant of SHP2's open-state stabilization. Mutations that disrupt the R111/E249 ion pair substantially shift SHP2's open/closed equilibrium to the closed state, even compared to wild-type SHP2's basal-state equilibrium, which strongly favors the closed state. To the best of our knowledge, the ion-pair variants uncovered in this study are the first known SHP2 mutants in which autoinhibition is augmented with respect to the wild-type protein. Such "hyperinhibited" mutants may provide useful tools for signaling studies that investigate the connections between SHP2 inhibition and the suppression of human disease progression.

Study on the allosteric activation mechanism of SHP2 via elastic network models and neural relational inference molecular dynamics simulation.

As a ubiquitous protein tyrosine phosphatase, SHP2 is involved in PD-1/PD-L1 mediated tumor immune escape and undergoes substantial conformational changes. Therefore, it is considered an ideal target for tumor intervention. However, the allosteric mechanisms of SHP2 binding PD-1 intracellular ITIM/ITSM phosphopeptides remain unclear, which greatly hinders the development of novel structure-based anticancer allosteric inhibitors. In this work, the open and closed structural models of SHP2 are first constructed based on this knowledge; next their motion modes are investigated via elastic network models such as the Gaussian network model (GNM), anisotropic network model (ANM) and adaptive anisotropic network model (aANM); and finally, a possible allosteric signaling pathway is proposed using a neural relational inference molecular dynamics (NRI-MD) simulation embedded with an artificial intelligence (AI) strategy. In GNM and ANM, the N-SH2, C-SH2 and PTP domains all exhibit distinct dynamics partitions, and the N-SH2/C-SH2 regions show a rigid rotation relative to PTP. According to a series of intermediate snapshots given by aANM, N-SH2 is first identified with pY223 specifically, inducing a D'E-loop to change from ß-sheets to random coils, and then, C-SH2 serves as a fulcrum to drive N-SH2 to rotate 110° completely away from the original active sites of PTP. Finally, a possible allosteric signaling-transfer path for SHP2, namely R220-R138-T108-R32, is proposed based on NRI-MD sampling. This work provides a possible allosteric mechanism of SHP2, which is helpful for the following design of novel allosteric inhibitors and is expected to be used in clinical synergies with PD-1 monoclonal antibody.

Allosteric modulation of SHP2: Quest from known to unknown.

Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) is a key regulatory factor in the cell cycle and its activating mutations play an important role in the development of various cancers, making it an important target for antitumor drugs. Due to the highly conserved amino acid sequence and positively charged nature of the active site of SHP2, it is difficult to discover inhibitors with high affinity for the catalytic site of SHP2 and sufficient cell permeability, making it considered an "undruggable" target. However, the discovery of allosteric regulation mechanisms provides new opportunities for transforming undruggable targets into druggable ones. Given the limitations of orthosteric inhibitors, SHP2 allosteric inhibitors have become a more selective and safer research direction. In this review, we elucidate the oncogenic mechanism of SHP2 and summarize the discovery methods of SHP2 allosteric inhibitors, providing new strategies for the design and improvement of SHP2 allosteric inhibitors.

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation.

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

Small-molecule Modulators Targeting SHP2 for Cancer Therapy.

BACKGROUND: SHP2 is a protein tyrosine phosphatase that is extensively involved in several signaling pathways related to cancer occurrence, and thus SHP2 has been proposed as an attractive target for cancer treatment. METHODS: After a brief introduction of SHP2, we provided a short overview of the structure, function and regulation mechanism of SHP2 in cancer occurrence. Then, this perspective focused on the current therapeutic strategies targeting SHP2, including SHP2 PTP inhibitors, SHP2 allosteric inhibitors and SHP2-targeting PROTACs, and discussed the benefits and defects of these strategies. Finally, the opportunities and challenges were presented. RESULTS: SHP2 regulated RAS-ERK, PI3K-AKT, JAK-STAT and PD-1/PD-L1 signaling pathways involved in the pathogenesis of cancer via conformations conversion. Current therapeutic strategies targeting SHP2, especially SHP2 allosteric inhibitors, hold significant potency and have broad application prospects for cancer therapy. CONCLUSION: In summary, SHP2 is a promising therapeutic target, and strategies targeting SHP2 offer an alternative program for cancer patients.

A comprehensive review of SHP2 and its role in cancer.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

Identification of the novel natural product inhibitors of SHP2 from the plant Toona sinensis: In vitro and in silico study.

In this study, we tested the inhibitory activity of 45 natural products extracted from the plant Toona sinensis on SHP2 protein, and identified four natural product inhibitors. The natural product 1,2,3,6-Tetragalloylglucose (A-1) was first reported as a competitive inhibitor of SHP2, with an IC50 value of 0.20 ± 0.029 µM and the selectivity of 1.8-fold and 4.35-fold to high homologous proteins SHP1 and PTP1B, respectively. Compound A-1 also showed high inhibitory activity on SHP2-E76K and SHP2-E76A mutants, with IC50 values of 0.95 ± 0.21 µM and 0.29 ± 0.045 µM, respectively. Cell viability assay showed that compound A-1 could inhibit the proliferation of a variety of cancer cells. Apoptosis assay showed that compound A-1 could effectively induce apoptosis of KRASG12C-mut NCI-H23 and KRASG12S-mut A549 cells. Western blot assay showed that compound A-1 could down regulate the phosphorylation levels of Erk1/2 and Akt in NCI-H23 and A549 cells. Molecular docking showed that compound A-1 could effectively dock to the catalytic active region of SHP2. Molecular dynamics simulation explored the effect of compound A-1 on SHP2, revealing the deep-seated binding mechanism. This study would provide valuable clues for the development of SHP2 and its mutant inhibitors.

Enhancing Adoptive Cell Therapy by T Cell Loading of SHP2 Inhibitor Nanocrystals before Infusion.

Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

Crystallographic landscape of SHP2 provides molecular insights for SHP2 targeted drug discovery.

The protein tyrosine phosphatase SHP2 encoded by PTPN11 is a promising therapeutic target for cancer therapy. The dynamic change of SHP2 between closed and open conformations under either physiological or pathological conditions provides opportunities to design SHP2 inhibitors for treating SHP2-related diseases. To date, several SHP2 allosteric inhibitors have advanced into clinical trials as mono- or combined therapy of cancers. In this review, we provide an overview on the structural landscape of SHP2 under physiological and pathological conditions and also comprehensively analyze the binding models of SHP2/inhibitor complexes. Structural features of SHP2 under pathological conditions and co-crystal structures of SHP2/inhibitor complexes will definitely facilitate structure-guided design of SHP2 inhibitors. Finally, proteolysis targeting chimeric (PROTAC) based SHP2 degraders have shown therapeutic promise for cancer therapy and are also briefly discussed. We hope this review could provide crystallographic landscape for SHP2 targeted drug discovery.

Double-edged roles of protein tyrosine phosphatase SHP2 in cancer and its inhibitors in clinical trials.

Phosphorylation is a reversible post-translational modification regulated by phosphorylase and dephosphorylase to mediate important cellular events. Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) encoded by PTPN11 is the first identified oncogenic protein in protein tyrosine phosphatases family. Serving as a convergent node, SHP2 is involved in multiple cascade signaling pathways including Ras-Raf-MEK-ERK, PI3K-AKT, JAK-STAT and PD-1/PD-L1 pathways. Especially, the double-edged roles of SHP2 based on the substrate specificity in various biological contexts dramatically increase the effect complexity in different SHP2-associated diseases. Evidences suggest that by collaborating with other mutations in associated pathways, dysregulation of SHP2 contributes to the pathogenesis of different cancers, making SHP2 a promising therapeutic target for cancer treatment. SHP2 can either act as oncogenic factor or tumor suppressor in different diseases, and both the conserved catalytic dephosphorylation mechanism and the unique allosteric regulation mechanism of SHP2 provide opportunities for the development of SHP2 inhibitors and activators. To date, several small-molecule SHP2 inhibitors have advanced into clinical trials for mono- or combined therapy of cancers. Moreover, SHP2 activators and proteolysis-targeting chimera (PROTAC)-based degraders also display therapeutic promise. In this review, we comprehensively summarize the overall structures, regulation mechanisms, double-edged roles of SHP2 in both physiological and carcinogenic pathways, and SHP2 inhibitors in clinical trials. SHP2 activators and degraders are also briefly discussed. This review aims to provide in-depth understanding of the biological roles of SHP2 and highlight therapeutic potential of targeting SHP2.

Exploring the cause of the dual allosteric targeted inhibition attaching to allosteric sites enhancing SHP2 inhibition.

SHP2 is a protein tyrosine phosphatase (PTP) that can regulate the tyrosine phosphorylation level. Overexpression of SHP2 will promote the development of cancer diseases, so SHP2 has become one of the popular targets for the treatment of cancer. Studies have reported that both SHP099 and SHP844 are inhibitors of SHP2 and bind to different allosteric sites 1 and 2, respectively. Studies have shown that combining SHP099 with SHP844 will enhance pharmacological pathway inhibition in cells. This study uses molecular dynamic simulations to explore the dual allosteric targeted inhibition mechanism. The result shows that the residues THR108-TRP112 (allosteric site 1) move to LEU236-GLN245 (αB-αC link loop in PTP domain) , the residues of GLN79-GLN87 (allosteric site 2) get close to LEU262-GLN269 (αA-αB link loop in PTP domain) and HIS458-ARG465 (P-loop) come near to ARG501-THR507 (Q-loop) in SHP2-SHP099-SHP844 system, which makes the "inactive conformation" more stable and prevents the substrate from entering the catalytic site. Meanwhile, residue GLU110 (allosteric site 1), ARG265 (allosteric site 2), and ARG501 (Q-loop) are speculated to be the key residues that causing the SHP2 protein in auto-inhibition conformation. It is hoped that this study will provide clues for the development of the dual allosteric targeted inhibition of SHP2.

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1.

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613-694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613-694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Determining folding and binding properties of the C-terminal SH2 domain of SHP2.

SH2 domains are a class of protein-protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras-ERK1/2 signaling pathway that possess two SH2 domains, namely, N-SH2 and C-SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the SH2 domains of SHP2 is Gab2, a scaffolding protein with critical role in determining cell differentiation. Despite their key biological role and the importance of a correct native fold to ensure it, the mechanism of binding of SH2 domains with their ligands and the determinants of their stability have been poorly characterized. In this article, we present a comprehensive kinetic study of the folding of the C-SH2 domain and the binding mechanism with a peptide mimicking a region of Gab2. Our data, obtained at different pH and ionic strength conditions and supported by site-directed mutagenesis, highlight the role of electrostatic interactions in the early events of recognition. Interestingly, our results suggest a key role of a highly conserved histidine residue among SH2 family in the interaction with negative charges carried by the phosphotyrosine of Gab2. Moreover, the analysis of the equilibrium and kinetic folding data of C-SH2 describes a complex mechanism implying a change in rate-limiting step at high denaturant concentrations. Our data are discussed under the light of previous works on N-SH2 domain of SHP2 and other SH2 domains.

Clinical variability of neurofibromatosis 1: A modifying role of cooccurring PTPN11 variants and atypical brain MRI findings.

Neurofibromatosis 1 (NF1) is a disorder characterized by variable expressivity caused by loss-of-function variants in NF1, encoding neurofibromin, a protein negatively controlling RAS signaling. We evaluated whether concurrent variation in proteins functionally linked to neurofibromin contribute to the variable expressivity of NF1. Parallel sequencing of a RASopathy gene panel in 138 individuals with molecularly confirmed clinical diagnosis of NF1 identified missense variants in PTPN11, encoding SHP2, a positive regulator of RAS signaling, in four subjects from three unrelated families. Three subjects were heterozygous for a gain-of-function variant and showed a severe expression of NF1 (developmental delay, multiple cerebral neoplasms and peculiar cortical MRI findings), and features resembling Noonan syndrome (a RASopathy caused by activating variants in PTPN11). Conversely, the fourth subject, who showed an attenuated presentation, carried a previously unreported PTPN11 variant that had a hypomorphic behavior in vitro. Our findings document that functionally relevant PTPN11 variants occur in a small but significant proportion of subjects with NF1 modulating disease presentation, suggesting a model in which the clinical expression of pathogenic NF1 variants is modified by concomitant dysregulation of protein(s) functionally linked to neurofibromin. We also suggest targeting of SHP2 function as an approach to treat evolutive complications of NF1.

A novel partially open state of SHP2 points to a "multiple gear" regulation mechanism.

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.

Exploring the Distinct Binding and Activation Mechanisms for Different CagA Oncoproteins and SHP2 by Molecular Dynamics Simulations.

CagA is a major virulence factor of Helicobacter pylori. H. pylori CagA is geographically subclassified into East Asian CagA and Western CagA, which are characterized by the presence of a EPIYA-D or EPIYA-C segment. The East Asian CagA is more closely associated with gastric cancer than the Western CagA. In this study, molecular dynamic (MD) simulations were performed to investigate the binding details of SHP2 and EPIYA segments, and to explore the allosteric regulation mechanism of SHP2. Our results show that the EPIYA-D has a stronger binding affinity to the N-SH2 domain of SHP2 than EPIYA-C. In addition, a single EPIYA-D binding to N-SH2 domain of SHP2 can cause a deflection of the key helix B, and the deflected helix B could squeeze the N-SH2 and PTP domains to break the autoinhibition pocket of SHP2. However, a single EPIYA-C binding to the N-SH2 domain of SHP2 cannot break the autoinhibition of SHP2 because the secondary structure of the key helix B is destroyed. However, the tandem EPIYA-C not only increases its binding affinity to SHP2, but also does not significantly break the secondary structure of the key helix B. Our study can help us better understand the mechanism of gastric cancer caused by Helicobacter pylori infection.

Exploring the dynamic mechanism of allosteric drug SHP099 inhibiting SHP2E69K.

The E69K mutation is one of the most frequent protein tyrosine phosphatase-2 (SHP2) mutations in leukemia, and it can cause the increase in the protein activity. Recent studies have shown that the E69K mutation was fairly sensitive to the allosteric inhibitor of SHP2 (SHP099). However, the molecular mechanism of the allosteric drug SHP099 inhibiting SHP2E69K remains unclear. Thus, the molecular dynamic simulations and the post-dynamics analyses (RMSF, PCA, DCCM, RIN and the binding free energies) for SHP2WT, SHP2WT-SHP099, SHP2E69K and SHP2E69K-SHP099 were carried out, respectively. Owing to the strong binding affinity of SHP099 to residues Thr219 and Arg220, the flexibility of linker region (residues Val209-Arg231) was reduced. Moreover, the presence of SHP099 kept the autoinhibition state of the SHP2 protein through enhancing the interactions between the linker region and Q loop in PTP domain, such as Thr219/Val490, Thr219/Asn491, Arg220/Ile488 and Leu254/Asn491. In addition, it was found that the residues (Thr219, Arg220, Leu254 and Asn491) might be the key residues responsible for the conformational changes of protein. Overall, this study may provide an important basis for understanding how the SHP099 effectively inhibited the SHP2E69K activity at the molecular level.