scholarly journals Crystal structure of death-associated protein kinase 1 in complex with the dietary compound resveratrol

IUCrJ ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. 131-138
Author(s):  
Takeshi Yokoyama ◽  
Ryoya Suzuki ◽  
Mineyuki Mizuguchi
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Kinase Domain ◽  
Crystallographic Analysis ◽  
Structural Basis ◽  
Atp Binding Site ◽  
Death Associated Protein Kinase

Death-associated protein kinase 1 (DAPK1) is a large multidomain protein with an N-terminal serine/threonine protein kinase domain. DAPK1 is considered to be a promising molecular target for the treatment of Alzheimer's disease (AD). In the present study, the inhibitory potency of resveratrol (RSV), a dietary polyphenol found in red wine, against the catalytic activity of DAPK1 was investigated. Kinetic and fluorescent probe competitive binding analyses revealed that RSV directly inhibited the catalytic activity of DAPK1 by binding to the ATP-binding site. Crystallographic analysis of DAPK1 in complex with RSV revealed that the A-ring of RSV occupied the nucleobase-binding position. Determination of the binding mode provided a structural basis for the design of more potent DAPK1 inhibitors. In conclusion, the data here clearly show that RSV is an ATP-competitive inhibitor of DAPK1, encouraging speculation that RSV may be useful for the development of AD inhibitors.

scholarly journals 14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Matej Horvath ◽  
Olivia Petrvalska ◽  
Petr Herman ◽  
Veronika Obsilova ◽  
Tomas Obsil
Keyword(s):  
Protein Kinase ◽  
Crystallographic Analysis ◽  
Structural Basis ◽  
Binding Motif ◽  
Scaffolding Proteins ◽  
Mode Iii ◽  
C Terminus ◽  
Motility Regulation ◽  
Death Associated Protein Kinase ◽  
Biophysical Techniques

AbstractDeath-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti‐inflammatory therapies.

Structure of mitogen-activated protein kinase kinase 1 in the DFG-out conformation

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Setsu Nakae ◽  
Maho Kitamura ◽  
Daisuke Fujiwara ◽  
Masaaki Sawa ◽  
Tsuyoshi Shirai ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Kinase ◽  
Binding Mode ◽  
Mitogen Activated Protein Kinase ◽  
Structural Elements ◽  
Atp Binding Site ◽  
Starting Point ◽  
Mitogen Activated Protein ◽  
Activation Segment ◽  
Protein Kinase Kinase

Eukaryotic protein kinases contain an Asp-Phe-Gly (DFG) motif, the conformation of which is involved in controlling the catalytic activity, at the N-terminus of the activation segment. The motif can be switched between active-state (DFG-in) and inactive-state (DFG-out) conformations: however, the mechanism of conformational change is poorly understood, partly because there are few reports of the DFG-out conformation. Here, a novel crystal structure of nonphosphorylated human mitogen-activated protein kinase kinase 1 (MEK1; amino acids 38–381) complexed with ATP-γS is reported in which MEK1 adopts the DFG-out conformation. The crystal structure revealed that the structural elements (the αC helix and HRD motif) surrounding the active site are involved in the formation/stabilization of the DFG-out conformation. The ATP-γS molecule was bound to the canonical ATP-binding site in a different binding mode that has never been found in previously determined crystal structures of MEK1. This novel ATP-γS binding mode provides a starting point for the design of high-affinity inhibitors of nonphosphorylated inactive MEK1 that adopts the DFG-out conformation.

Metal coordination in kinases and pseudokinases

2017 ◽  
Vol 45 (3) ◽  
pp. 653-663 ◽  
Author(s):  
Matthias J. Knape ◽  
Friedrich W. Herberg
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Metal Binding ◽  
Binding Mode ◽  
Structural Basis ◽  
Phosphoryl Transfer ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Divalent Metal Ions ◽  
Functional Roles

Protein phosphorylation, mediated by protein kinases, is a key event in the regulation of eukaryotic signal transduction. The majority of eukaryotic protein kinases perform phosphoryl transfer, assisted by two divalent metal ions. About 10% of all human protein kinases are, however, thought to be catalytically inactive. These kinases lack conserved residues of the kinase core and are classified as pseudokinases. Yet, it has been demonstrated that pseudokinases are critically involved in biological functions. Here, we show how pseudokinases have developed strategies by modifying amino acid residues in order to achieve stable, active-like conformations. This includes binding of the co-substrate ATP in a two metal-, one metal- or even no metal-binding mode. Examples of the respective pseudokinases are provided on a structural basis and compared with a canonical protein kinase, Protein Kinase A. Moreover, the functional roles of both independent metal-binding sites, Me1 and Me2, are discussed. Lack of phosphotransferase activity does not implicate a loss of function and can easily point to alternative roles of pseudokinases, i.e. acting as switches or scaffolds, and having evolved as components crucial for cellular cross-talk and signaling. Interestingly, pseudokinases are present in all kingdoms of life and their specific roles remain enigmatic. More studies are needed to unravel the crucial functions of those interesting proteins.

scholarly journals Identification of CDK7 Inhibitors from Natural Sources Using Pharmacoinformatics and Molecular Dynamics Simulations

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1197
Author(s):  
Vikas Kumar ◽  
Shraddha Parate ◽  
Gunjan Thakur ◽  
Gihwan Lee ◽  
Hyeon-Su Ro ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Binding Affinity ◽  
Cost Effective ◽  
Binding Mode ◽  
Kinase Domain ◽  
Dynamics Simulation ◽  
Mode Analysis ◽  
Atp Binding Site ◽  
Dynamics Simulations

The cyclin-dependent kinase 7 (CDK7) plays a crucial role in regulating the cell cycle and RNA polymerase-based transcription. Overexpression of this kinase is linked with various cancers in humans due to its dual involvement in cell development. Furthermore, emerging evidence has revealed that inhibiting CDK7 has anti-cancer effects, driving the development of novel and more cost-effective inhibitors with enhanced selectivity for CDK7 over other CDKs. In the present investigation, a pharmacophore-based approach was utilized to identify potential hit compounds against CDK7. The generated pharmacophore models were validated and used as 3D queries to screen 55,578 natural drug-like compounds. The obtained compounds were then subjected to molecular docking and molecular dynamics simulations to predict their binding mode with CDK7. The molecular dynamics simulation trajectories were subsequently used to calculate binding affinity, revealing four hits—ZINC20392430, SN00112175, SN00004718, and SN00262261—having a better binding affinity towards CDK7 than the reference inhibitors (CT7001 and THZ1). The binding mode analysis displayed hydrogen bond interactions with the hinge region residues Met94 and Glu95, DFG motif residue Asp155, ATP-binding site residues Thr96, Asp97, and Gln141, and quintessential residue outside the kinase domain, Cys312 of CDK7. The in silico selectivity of the hits was further checked by docking with CDK2, the close homolog structure of CDK7. Additionally, the detailed pharmacokinetic properties were predicted, revealing that our hits have better properties than established CDK7 inhibitors CT7001 and THZ1. Hence, we argue that proposed hits may be crucial against CDK7-related malignancies.

Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms

1989 ◽  
Vol 9 (7) ◽  
pp. 2890-2896
Author(s):  
J R Downing ◽  
M F Roussel ◽  
C J Sherr
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ligand Binding ◽  
Kinase Domain ◽  
Colony Stimulating Factor ◽  
Proteolytic Fragment ◽  
Atp Binding Site ◽  
Kinase C ◽  
Phosphopeptide Mapping ◽  
Stimulating Factor

The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.

scholarly journals Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition

2003 ◽  
Vol 375 (2) ◽  
pp. 255-262 ◽  
Author(s):  
David KOMANDER ◽  
Gursant S. KULAR ◽  
Jennifer BAIN ◽  
Matthew ELLIOTT ◽  
Dario R. ALESSI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hydroxy Group ◽  
Active Site ◽  
Binding Pocket ◽  
Kinase Domain ◽  
Structural Basis ◽  
Active Site Residues ◽  
Dependent Protein Kinase ◽  
Growth Factor Signalling ◽  
Dependent Protein

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.

AtCERK1 Phosphorylation Site S493 Contributes to the Transphosphorylation of Downstream Components for Chitin-Induced Immune Signaling

2019 ◽  
Vol 60 (8) ◽  
pp. 1804-1810 ◽  
Author(s):  
Maruya Suzuki ◽  
Issei Yoshida ◽  
Kenkichi Suto ◽  
Yoshitake Desaki ◽  
Naoto Shibuya ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Induced Defense ◽  
Phosphorylation Site ◽  
Defense Responses ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Structural Basis ◽  
Downstream Signaling ◽  
Signaling Components

Abstract While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

Structural and thermodynamic analyses of interactions between death-associated protein kinase 1 and anthraquinones

2020 ◽  
Vol 76 (5) ◽  
pp. 438-446 ◽  
Author(s):  
Takeshi Yokoyama ◽  
Peter Wijaya ◽  
Yuto Kosaka ◽  
Mineyuki Mizuguchi
Keyword(s):  
Protein Kinase ◽  
Amyloid Β ◽  
Crystallographic Analysis ◽  
Binding Affinities ◽  
Hydrogen Bond Network ◽  
Lead Compound ◽  
Death Associated Protein Kinase ◽  
Bond Network ◽  
Inhibition Potencies ◽  
Potent Inhibitory Activity

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase that regulates apoptosis and autophagy. DAPK1 is considered to be a therapeutic target for amyloid-β deposition, endometrial adenocarcinomas and acute ischemic stroke. Here, the potent inhibitory activity of the natural anthraquinone purpurin against DAPK1 phosphorylation is shown. Thermodynamic analysis revealed that while the binding affinity of purpurin is similar to that of CPR005231, which is a DAPK1 inhibitor with an imidazopyridazine moiety, the binding of purpurin was more enthalpically favorable. In addition, the inhibition potencies were correlated with the enthalpic changes but not with the binding affinities. Crystallographic analysis of the DAPK1–purpurin complex revealed that the formation of a hydrogen-bond network is likely to contribute to the favorable enthalpic changes and that stabilization of the glycine-rich loop may cause less favorable entropic changes. The present findings indicate that purpurin may be a good lead compound for the discovery of inhibitors of DAPK1, and the observation of enthalpic changes could provide important clues for drug development.

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