Glycine substitution in SH3-SH2 connector of Hck tyrosine kinase causes population shift from assembled to disassembled state.

A combination of small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations based on a coarse grained model is used to examine the effect of glycine substitutions in the short connector between the SH3 and SH2 domains of Hck, a member of the Src-family kinases. It has been shown previously that the activity of cSrc kinase is upregulated by substitution of 3 residues by glycine in the SH3-SH2 connector. Here, analysis of SAXS data indicates that the population of Hck in the disassembled state increases from 25% in the wild type kinase to 76% in the glycine mutant. This is consistent with the results of free energy perturbation calculations showing that the mutation in the connector shifts the equilibrium from the assembled to the disassembled state. This study supports the notion that the SH3-SH2 connector helps to regulate the activity of tyrosine kinases by shifting the population of the active state of the multidomain protein independent of C-terminal phosphorylation.

Targeting Small Molecule Tyrosine Kinases by Polyphenols: New Move Towards Anti-tumor Drug Discovery.

BACKGROUND: Cancer is a complex disease involving genetic and epigenetic alteration that allows cells to escape normal homeostasis. Kinases play a crucial role in signaling pathways that regulate cell functions. Deregulation of kinases leads to a variety of pathological changes, activating cancer cell proliferation and metastases. The molecular mechanism of cancer is complex and the dysregulation of tyrosine kinases like Anaplastic Lymphoma Kinase (ALK), Bcr-Abl (Fusion gene found in patient with Chronic Myelogenous Leukemia (CML), JAK (Janus Activated Kinase), Src Family Kinases (SFKs), ALK (Anaplastic lymphoma Kinase), c-MET (Mesenchymal- Epithelial Transition), EGFR (Epidermal Growth Factor receptor), PDGFR (Platelet-Derived Growth Factor Receptor), RET (Rearranged during Transfection) and VEGFR (Vascular Endothelial Growth Factor Receptor) plays major role in the process of carcinogenesis. Recently, kinase inhibitors have overcome many problems of traditional cancer chemotherapy as they effectively separate out normal, non-cancer cells as well as rapidly multiplying cancer cells. METHODS: Electronic databases were searched to explore the small molecule tyrosine kinases by polyphenols with the help of docking study (Glide-7.6 program interfaced with Maestro-v11.3 of Schrödinger 2017) to show the binding energies of polyphenols inhibitor with different tyrosine kinases in order to differentiate between the targets. RESULTS: From the literature survey, it was observed that the number of polyphenols derived from natural sources alters the expression and signaling cascade of tyrosine kinase in various tumor models. Therefore, the development of polyphenols as a tyrosine kinase inhibitor against targeted proteins is regarded as an upcoming trend for chemoprevention. CONCLUSION: In this review, we have discussed the role of polyphenols as chemoreceptive which will help in future for the development and discovery of novel semisynthetic anticancer agents coupled with polyphenols.

A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region.

Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region.

Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (super)families, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest), reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs)-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human tyrosine kinase family, using all available kinase catalytic domain structures from any organism as structural templates. Ensembler is free and open source software licensed under the GNU General Public License (GPL) v2. It is compatible with Linux and OS X. The latest release can be installed via the conda package manager, and the latest source can be downloaded from https://github.com/choderalab/ensembler.

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Atomistic view of the conformational activation of Src kinase using the string method with swarms-of-trajectories.

The inactive-to-active conformational transition of the catalytic domain of human c-Src tyrosine kinase is characterized using the string method with swarms-of-trajectories with all-atom explicit solvent molecular dynamics simulations. The activation process occurs in two main steps in which the activation loop (A-loop) opens first, followed by the rotation of the alphaC helix. The computed potential of mean force energy along the activation pathway displays a local minimum, which allows the identification of an intermediate state. These results show that the string method with swarms-of-trajectories is an effective technique to characterize complex and slow conformational transitions in large biomolecular systems.

Conformational rearrangements upon Syk auto-phosphorylation.

Syk is a cytoplasmic tyrosine kinase that is activated after recruitment to immune receptors, triggering the phopshorylation of downstream targets. The kinase activity of Syk is controlled by an auto-inhibited conformation consisting of a regulatory region that contains two N-terminal Src homology 2 (SH2) domains inhibiting the catalytic activity of the kinase domain located at the C-terminus. The atomic structure of the related Zap-70 kinase and an electron microscopy (EM) model of Syk have revealed the structural mechanism of this auto-inhibition based on the formation of a compact conformation sustained by interactions between the regulatory and catalytic domains. On the other hand, the structural basis of Syk activation is not fully understood due to the lack of a 3D structure of full-length Syk in an active conformation. Here, we have used single-particle electron microscopy to analyse the conformational changes taking place in an activated form of Syk induced by auto-phosphorylation. The conformation of phosphorylated Syk is reminiscent of the compact structure of the inhibited protein but significant conformational changes are observed in the regulatory region. These rearrangements could be sufficient to disrupt the inhibitory interactions, contributing to Syk activation. These results suggest that the regulation of the activation of Syk might be modulated by subtle changes in the positioning of the regulatory domains rather than a full opening mechanism as proposed for the Src kinases.

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor.

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor.

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.

Leveraging structural approaches: applications of NMR-based screening and X-ray crystallography for inhibitor design.

In the last several years, NMR strategies in drug discovery have evolved from a primarily structural focus to a set of technologies that are non-structural in nature but that have a much greater impact on the identification and optimization of real drug leads. NMR-based screening methods, such as the SHAPES strategy, help rapidly identify good starting points for drug design in a relatively high throughput implementation. The SHAPES method uses simple NMR techniques to detect binding of a limited, but diverse library of low molecular weight, soluble compounds to a potential drug target. SHAPES library compounds are derived largely from molecular frameworks most commonly found in known therapeutic agents. The NMR experiments used in these protocols are based on the well-known NMR techniques, and may be applied to targets with no limitation on molecular weight and no requirement for isotope labeling. Following screening, SHAPES hits may be used to guide virtual screening, synthesis of combinatorial libraries, and bias the first compounds that undergo high throughput screening. Integration of the SHAPES strategy with iterative X-ray crystallographic structure determination can be very useful in deriving an initial structural pharmacophore model and achieving significant in vitro potency in a short time frame. Here, examples are provided of how the combination of NMR SHAPES screening, virtual screening, molecular modeling and X-ray crystallography has led to novel drug scaffolds in several drug discovery programs: JNK3 MAP kinase and the fatty acid binding protein, aP2.

Lyn and Syk kinases are sequentially engaged in phagocytosis mediated by Fc gamma R.

Recent data indicate that phagocytosis mediated by FcgammaRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcgammaRs (4 degrees C) were accompanied by high amounts of Lyn, in addition to the signaling gamma-chain of FcgammaRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37 degrees C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the gamma-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the gamma-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcgammaRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.

Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation.

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.

Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.

The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.

Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation.

To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT). In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state. Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state. Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity. These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor. Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele.

Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.