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Your search keyword '"Handa, Noriko"' showing total 3 results
Start Over Author "Handa, Noriko" Topic adenosine triphosphate
3 results on '"Handa, Noriko"'

1. Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002.

Author

Parker LJ, Taruya S, Tsuganezawa K, Ogawa N, Mikuni J, Honda K, Tomabechi Y, Handa N, Shirouzu M, Yokoyama S, and Tanaka A

Subjects
AMP-Activated Protein Kinases antagonists & inhibitors, AMP-Activated Protein Kinases chemistry, AMP-Activated Protein Kinases genetics, Binding, Competitive, Crystallization, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli metabolism, High-Throughput Screening Assays, Humans, Ligands, MAP Kinase Kinase Kinase 5 antagonists & inhibitors, MAP Kinase Kinase Kinase 5 chemistry, MAP Kinase Kinase Kinase 5 genetics, Mitogen-Activated Protein Kinase 14 antagonists & inhibitors, Mitogen-Activated Protein Kinase 14 chemistry, Mitogen-Activated Protein Kinase 14 genetics, Protein Binding, Proto-Oncogene Proteins c-hck antagonists & inhibitors, Proto-Oncogene Proteins c-hck chemistry, Proto-Oncogene Proteins c-hck genetics, Proto-Oncogene Proteins c-pim-1 antagonists & inhibitors, Proto-Oncogene Proteins c-pim-1 chemistry, Proto-Oncogene Proteins c-pim-1 genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Adenosine Triphosphate chemistry, Fluorescent Dyes chemistry, Imidazoles chemistry, Molecular Probes chemistry, Protein Kinase Inhibitors chemistry, Thiazoles chemistry
Abstract

The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.

Published
2014
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2. The C-terminal BAG domain of BAG5 induces conformational changes of the Hsp70 nucleotide-binding domain for ADP-ATP exchange.

Author

Arakawa A, Handa N, Ohsawa N, Shida M, Kigawa T, Hayashi F, Shirouzu M, and Yokoyama S

Subjects
Adaptor Proteins, Signal Transducing, Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Binding Sites, Carrier Proteins metabolism, Crystallography, X-Ray, HSP70 Heat-Shock Proteins metabolism, Humans, Protein Structure, Tertiary, Adenosine Diphosphate chemistry, Adenosine Triphosphate chemistry, Carrier Proteins chemistry, HSP70 Heat-Shock Proteins chemistry
Abstract

ADP-ATP exchange by the molecular chaperone Hsp70 is enhanced by several cochaperones. BAG5 consists of five BAG domains and associates with the nucleotide-binding domain (NBD) of Hsp70. The overexpression of BAG5 in the cytosol reportedly disturbs Hsp70-mediated protein refolding and induces Parkinson's disease. In the present study, we found that the fifth BAG domain (BD5) of BAG5 is responsible for the interaction between Hsp70 and BAG5. We also determined the crystal structures of the BD5*NBD complex. BD5 binding caused two different types of NBD conformational changes, which both disrupted the nucleotide-binding groove. In fact, BD5 reduced the affinity of the NBD for ADP. Moreover, BD5, as well as the full-length BAG5, accelerated Hsp70-mediated refolding in an in vitro assay. Therefore, BAG5 can function as the nucleotide exchange factor of Hsp70 for the enhancement of protein refolding.

Published
2010
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3. Biochemical and structural studies on the high affinity of Hsp70 for ADP

Author

Arakawa, Akihiko, Handa, Noriko, Shirouzu, Mikako, and Yokoyama, Shigeyuki

Subjects
Adenosine Diphosphate, Adenosine Triphosphate, Binding Sites, Molecular Sequence Data, Escherichia coli, Humans, HSP70 Heat-Shock Proteins, Magnesium, Amino Acid Sequence, Crystallography, X-Ray, Sequence Alignment, Article, Recombinant Proteins
Abstract

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the β-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.

Published
2011

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