scholarly journals A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

2020 ◽  
Vol 295 (11) ◽  
pp. 3563-3575 ◽  
Author(s):  
Zachary Hartman ◽  
Werner J. Geldenhuys ◽  
Yehenew M. Agazie
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Specific Binding ◽  
Tyrosine Phosphatase ◽  
Strong Inhibitor ◽  
Selective Binding ◽  
Specific Amino Acid ◽  
Src Homology ◽  
Her2 Signaling

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1–4 or the human homologs HER1–4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase

1994 ◽  
Vol 14 (1) ◽  
pp. 509-517
Author(s):  
W Li ◽  
R Nishimura ◽  
A Kashishian ◽  
A G Batzer ◽  
W J Kim ◽  
...  
Keyword(s):  
Growth Factor ◽  
Intracellular Signaling ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Growth Factor Receptors ◽  
Ras Signaling ◽  
Pdgf Receptor ◽  
Phosphotyrosine Phosphatase ◽  
Src Homology

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.

scholarly journals Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

2018 ◽  
Vol 116 (2) ◽  
pp. 679-688 ◽  
Author(s):  
Ming-ling Liao ◽  
George N. Somero ◽  
Yun-wei Dong
Keyword(s):  
Amino Acid ◽  
Thermal Adaptation ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Amino Acid Substitutions ◽  
Thermal Stabilities ◽  
Specific Amino Acid ◽  
Enzyme Thermal Stability ◽  
And Function

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

scholarly journals Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

2016 ◽  
Author(s):  
Wesley G. Chen ◽  
Jacob Witten ◽  
Scott C. Grindy ◽  
Niels Holten-Andersen ◽  
Katharina Ribbeck
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Amino Acid Sequences ◽  
Nuclear Pore ◽  
Selective Binding ◽  
Charged Amino Acids ◽  
Essential Components ◽  
Transport Receptors

AbstractThe nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

PSD-93 mediate the dialogue between neuron and microglia and facilitate acute ischemic stroke by binding 357-395 amino acid sequence of CX3CL1

2020 ◽  
Author(s):  
Qingxiu Zhang ◽  
Lei He ◽  
Mo Chen ◽  
Hui Yang ◽  
Xiaowei Cao ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ischemia Reperfusion ◽  
Yeast Two Hybrid ◽  
Neuron Death ◽  
Specific Amino Acid ◽  
Ischemic Brain ◽  
Two Hybrid

Abstract Background: Our previous experiments demonstrated that PSD-93 mediates glutamate excitotoxicity induced by ischemic brain injury, which promotes the release of inflammatory cytokines in early ischemic brain injury by activating the NMDA receptor. Glutamate activity is the key to neuronal excitatory toxicity and microglial cell inflammatory response in the joints. However, the underlying mechanisms of how does PSD-93 mediate the dialogue between neurons and microglia in the postsynaptic dense region remain elusive. And CX3 chemokine ligand 1 (CX3CL1) is a chemokine that is specifically expressed in neurons. Its only receptor CX3CR1 is highly expressed in microglia and its main forms are membrane binding and soluble. In this study, we aim to clarify the specific amino acid sequence of the binding of psd-93 and CX3CL1 and investigate role of PSD-93 on regulating the crosstalk between neuron and microglia in acute ischemic stroke. Methods: In this study, male C57BL/6 mice aged 8-12 weeks and weighted 22-26g were applied with Middle Cerebral Artery Occlusion (MCAO) model and randomly divided into different groups. Firstly, co-immunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points 3h, 6h, 12h 24h, 48h and 72h following cerebral ischemic/reperfusion. Meanwhile, ELISA was used to investigate the expression of soluble CX3CL1 at the same time points to confirm the relationship between of the expression of soluble CX3CL1 and the combination of PSD-93 and CX3CL1. Secondly, two bait plasmids pSos-PSD-93-full length, pSos-CX3CL1-full length and five mutant plasmids: pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4, and pMyr-CX3CL1-mut, were constructed and used a yeast two-hybrid system to screen and identify positive clones and to determine the sequence in which the two proteins bind to each other. Thirdly, the proteins corresponding to the three positive clones obtained in the yeast two-hybrid experiment were used to construct plasmids for transfection of eukaryotic cells and the protein expression binding was verified again by in vitro co-immunoprecipitation. Finally, a specific fusion small peptide Tat-CX3CL1 were designed according to above experiment to inhibit the integration of PSD-93 and CX3CL1 and to explore their role on neuron death following reperfusion. Results: We found that the binding capacity of PSD-93 and CX3CL1 proteins peaked at 6h after ischemia/reperfusion and then decreased gradually. The specific amino acid sequence of PSD-93 and CX3CL1 binding was obtained by yeast double hybridization and in vitro immunoprecipitation. We identified that their binding sites are located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. And a specific fusion small peptide Tat-CX3CL1 (357-395aa) were designed to inhibit the integration of PSD-93 and CX3CL1 and perform neuroprotection on neuron death following reperfusion. Conclusions: Our results suggest that PSD-93 promotes the formation of its soluble form by binding to CX3CL1, which is recruited to the surface of microglia to bind to CX3CR1, thereby activating microglia to initiate inflammation. Thus, specific blockade of PSD-93-CX3CL1 coupling can reduce ischemia-reperfusion induced neuronal cell death, which provide a new target to treat ischemic stroke.

scholarly journals Leishmania EF-1α Activates the Src Homology 2 Domain Containing Tyrosine Phosphatase SHP-1 Leading to Macrophage Deactivation

2002 ◽  
Vol 277 (51) ◽  
pp. 50190-50197 ◽  
Author(s):  
Devki Nandan ◽  
Taolin Yi ◽  
Martin Lopez ◽  
Crystal Lai ◽  
Neil E. Reiner
Keyword(s):  
Leishmania Donovani ◽  
Tyrosine Phosphatase ◽  
Elongation Factor ◽  
Host Protein ◽  
Src Homology 2 ◽  
Infected Cells ◽  
Src Homology ◽  
Src Homology 2 Domain

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genusLeishmania.The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates ofLeishmania donovanipromastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification ofLeishmaniaelongation factor-1α (EF-1α) as a SHP-1-binding protein. PurifiedLeishmaniaEF-1α, but not host cell EF-1α, bound directly to SHP-1in vitroleading to its activation. Three independent lines of evidence indicated thatLeishmaniaEF-1α may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [35S]methionine-labeled organisms containedLeishmaniaEF-1α. Second, confocal, fluorescence microscopy usingLeishmania-specific antisera detectedLeishmaniaEF-1α in the cytosol of infected cells. Third, co-immunoprecipitation showed thatLeishmaniaEF-1α was associated with SHP-1in vivoin infected cells. Finally, introduction of purifiedLeishmaniaEF-1α, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-γ. Thus,LeishmaniaEF-1α is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.

scholarly journals Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

2015 ◽  
Vol 112 (20) ◽  
pp. 6347-6352 ◽  
Author(s):  
Bradley M. Hover ◽  
Nam K. Tonthat ◽  
Maria A. Schumacher ◽  
Kenichi Yokoyama
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
De Novo ◽  
Specific Binding ◽  
Molybdenum Cofactor ◽  
Enzyme Active Site ◽  
Complex Rearrangement ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH2GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

scholarly journals Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet

1998 ◽  
Vol 180 (14) ◽  
pp. 3563-3569 ◽  
Author(s):  
Jeryl L. Appleby ◽  
Robert B. Bourret
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Response Regulator ◽  
Hydroxyl Group ◽  
Amino Acid Substitutions ◽  
Mutant Proteins ◽  
Hydroxy Amino Acid ◽  
Hydroxy Amino ◽  
Flagellar Rotation

ABSTRACT CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

scholarly journals Small molecule control of neurotransmitter sulfonation

2020 ◽  
pp. jbc.RA120.015177
Author(s):  
Ian Cook ◽  
Mary Cacace ◽  
Ting Wang ◽  
Kristie Darrah ◽  
Alexander Deiters ◽  
...  
Keyword(s):  
Active Site ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Specific Binding ◽  
Protein A ◽  
Binding Pocket ◽  
Human Mammary Epithelial Cell ◽  
Primary Objective ◽  
Epithelial Cell Line

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder — a disease that afflicts ~20% of the world’s population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. Toward this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3) — the isoform responsible for sulfonating ~80% of the serotonin in extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a sidechain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization — the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable to those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

scholarly journals Molecular Dynamics Simulations of A27S and K120A Mutated PTP1B Reveals Selective Binding of the Bidentate Inhibitor

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Xi Chen ◽  
Xia Liu ◽  
Qiang Gan ◽  
Changgen Feng ◽  
Qian Zhang
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Binding Site ◽  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Tyrosine Phosphatase ◽  
Selective Binding ◽  
Protein Tyrosine ◽  
Ptp1b Inhibitors ◽  
Dynamics Simulations

Protein tyrosine phosphatase 1B (PTP1B) is considered a potential target for the treatment of type II diabetes and obesity due to its critical negative role in the insulin signaling pathway. However, improving the selectivity of PTP1B inhibitors over the most closely related T-cell protein tyrosine phosphatase (TCPTP) remains a major challenge for inhibitor development. Lys120 at the active site and Ser27 at the second pTyr binding site are distinct in PTP1B and TCPTP, which may bring differences in binding affinity. To explore the determinant of selective binding of inhibitor, molecular dynamics simulations with binding free energy calculations were performed on K120A and A27S mutated PTP1B, and the internal changes induced by mutations were investigated. Results reveal that the presence of Lys120 induces a conformational change in the WPD-loop and YRD-motif and has a certain effect on the selective binding at the active site. Ser27 weakens the stability of the inhibitor at the second pTyr binding site by altering the orientation of the Arg24 and Arg254 side chains via hydrogen bonds. Further comparison of alanine scanning demonstrates that the reduction in the energy contribution of Arg254 caused by A27S mutation leads to a different inhibitory activity. These observations provide novel insights into the selective binding mechanism of PTP1B inhibitors to TCPTP.

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