The role of water molecules in the binding of class I and II peptides to the SH3 domain of the Fyn tyrosine kinase

Interactions of proline-rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein–protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high-affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation–π and salt-bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family.

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Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7708-7717.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7708-7717

Author(s):

K V Prasad ◽

R Kapeller ◽

O Janssen ◽

H Repke ◽

J S Duke-Cohan ◽

...

Keyword(s):

T Cells ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Sh3 Domain ◽

Sh2 Domain ◽

Activation Process ◽

Lipid Kinase ◽

Sh3 Domains ◽

Protein Tyrosine ◽

Hiv 1

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

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The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study

Journal of Computer-Aided Molecular Design ◽

10.1007/s10822-019-00224-w ◽

2019 ◽

Vol 33 (9) ◽

pp. 787-797 ◽

Cited By ~ 1

Author(s):

Zoltán Orgován ◽

György G. Ferenczy ◽

György M. Keserű

Keyword(s):

Virtual Screening ◽

Ligand Binding ◽

Conformational Changes ◽

Metabotropic Glutamate Receptor ◽

Protein Structures ◽

Protein Flexibility ◽

Water Molecules ◽

Large Set ◽

Allosteric Modulators

Abstract Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

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The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

Biochemical Journal ◽

10.1042/bj3330811 ◽

1998 ◽

Vol 333 (3) ◽

pp. 811-816 ◽

Cited By ~ 14

Author(s):

Antonio PÁRRAGA ◽

Isabel GARCÍA-SÁEZ ◽

Sinead B. WALSH ◽

Timothy J. MANTLE ◽

Miquel COLL

Keyword(s):

Conformational Changes ◽

Active Site ◽

Three Dimensional ◽

Dimensional Structure ◽

Water Molecules ◽

X Ray Diffraction ◽

Glutathione S Transferase ◽

X Ray ◽

The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

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The Role of Hypoxia and SRC Tyrosine Kinase in Glioblastoma Invasiveness and Radioresistance

Cancers ◽

10.3390/cancers12102860 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2860 ◽

Cited By ~ 1

Author(s):

Filippo Torrisi ◽

Nunzio Vicario ◽

Federica M. Spitale ◽

Francesco P. Cammarata ◽

Luigi Minafra ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Poor Prognosis ◽

High Rate ◽

Crucial Importance ◽

Src Tyrosine Kinase ◽

Complete Surgical Resection ◽

Radiation Induced ◽

Proliferation And Invasion

Advances in functional imaging are supporting neurosurgery and radiotherapy for glioblastoma, which still remains the most aggressive brain tumor with poor prognosis. The typical infiltration pattern of glioblastoma, which impedes a complete surgical resection, is coupled with a high rate of invasiveness and radioresistance, thus further limiting efficient therapy, leading to inevitable and fatal recurrences. Hypoxia is of crucial importance in gliomagenesis and, besides reducing radiotherapy efficacy, also induces cellular and molecular mediators that foster proliferation and invasion. In this review, we aimed at analyzing the biological mechanism of glioblastoma invasiveness and radioresistance in hypoxic niches of glioblastoma. We also discussed the link between hypoxia and radiation-induced radioresistance with activation of SRC proto-oncogene non-receptor tyrosine kinase, prospecting potential strategies to overcome the current limitation in glioblastoma treatment.

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Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector

Biochemical Journal ◽

10.1042/bj3100049 ◽

1995 ◽

Vol 310 (1) ◽

pp. 49-53 ◽

Cited By ~ 3

Author(s):

R De Cristofaro ◽

M Picozzi ◽

E De Candia ◽

B Rocca ◽

R Landolfi

Keyword(s):

Conformational Changes ◽

Potential Role ◽

Competitive Inhibition ◽

Entropy Change ◽

Bulk Water ◽

Water Molecules ◽

Rabbit Lung ◽

Allosteric Effector ◽

Free Energy Of Binding

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.

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Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2883 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2883-2894 ◽

Cited By ~ 127

Author(s):

B J Mayer ◽

D Baltimore

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Sh3 Domains ◽

Sh2 Domains ◽

Abl Tyrosine Kinase ◽

Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

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Interaction of Guanylyl Cyclase C with SH3 Domain of Src Tyrosine Kinase

Journal of Biological Chemistry ◽

10.1074/jbc.m301153200 ◽

2003 ◽

Vol 278 (27) ◽

pp. 24342-24349 ◽

Cited By ~ 2

Author(s):

Rita Singh

Keyword(s):

Tyrosine Kinase ◽

Guanylyl Cyclase ◽

Sh3 Domain ◽

Guanylyl Cyclase C ◽

Src Tyrosine Kinase

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p130Cas mediates the transforming properties of the anaplastic lymphoma kinase

Blood ◽

10.1182/blood-2005-03-1204 ◽

2005 ◽

Vol 106 (12) ◽

pp. 3907-3916 ◽

Cited By ~ 42

Author(s):

Chiara Ambrogio ◽

Claudia Voena ◽

Andrea D. Manazza ◽

Roberto Piva ◽

Ludovica Riera ◽

...

Keyword(s):

Tyrosine Kinase ◽

Anaplastic Lymphoma Kinase ◽

Kinase Activity ◽

Adaptor Protein ◽

Dominant Negative ◽

Lymphoid Cells ◽

Src Tyrosine Kinase ◽

Anaplastic Lymphoma ◽

Proteomic Approach

Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)–ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry–based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor–bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALKK210R or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

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