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Neutron crystal structure analysis of human casein kinase II

Vendredi 8 mars 11:00 - Duree : 1 heure
Lieu : Science Building Room 036 - EPN campus - 71 avenue des Martyrs - Grenoble

Orateur : Motoyasu ADACHI (National Institutes for Quantum and Radiological Science and Technology, Japan)

Casein kinase II (CK2) is a serine/threonine kinase ubiquitously distributed among eukaryotic cells, and is known to be involved in the cell cycle and cell survival and proliferation. CK2 is one of the drug target proteins, because the relationship between CK2 over-expression and carcinogenesis and cancer metastasis has been pointed out. We aim to obtain the information for location of the hydrogen atoms and hydration structure, and to elucidate the catalytic reaction of CK2 for development of effective inhibitors. At the first step, we solved the structure by using neutron diffraction data for wild type CK2 without ligand [1].

Recombinant and hydrogenated CK2α of catalytic subunit was produced using an E. coli expression system and purified by column chromatography. For neutron diffraction experiments, large crystals in size of about 2 mm3 were prepared by the macro seeding method. After the dialysis for an obtained crystal against deuterium solvent, we collected a neutron diffraction data by FRMII BioDIFF at 100 K to 1.90 Å resolution. X-Ray diffraction data were collected at KEK-PF at BL-5A beam line for X-ray neutron joint refinement.

The crystal structure including hydrogen and deuterium atoms was refined by joint refinement to 1.9 and 1.1 Å resolution for neutron and X-ray diffraction data, respectively. Interestingly, a long hydrogen bonding network was found passing through inside of the protein from catalytic site to the other side of the protein based on the observation for exchanged deuterium atoms. His148 is highly conserved among serine/threonine kinases, and forms a part of the long hydrogen bonding relay with Asp214. Therefore, we prepared the CK2 mutants that substitute His148 with Ala, Ser and Asn. As a result, the kinase activity of the CK2 mutants significantly decreased as compared with the wild type. These results indicate that the hydrogen bonding network plays an important role in the enzymatic reaction of CK2. This information will contribute to the development of an anticancer drug with another inhibitory mechanism.

In addition to the neutron crystal structure analysis of human casein kinase II, I would like to mention protein perdeuteration for crystallography with our recent research for T4 lysozyme, and focus on the methyl configuration in proteins for this seminar. The observations may provide a new insight into the analyses for protein dynamics and by quantum chemical calculations.

[1] Shibazaki C, Arai S, Shimizu R, Saeki M, Kinoshita T, Ostermann A, Schrader TE, Kurosaki Y, Sunami T, Kuroki R, Adachi M. Hydration structures of the human protein kinase CK2α clarified by joint neutron and X-ray crystallography ; J. Mol. Biol. 430(24), 5094-5104 (2018).

Contact : mader@ill.fr

Discipline évènement : (Physique)
Entité organisatrice : (ILL)
Nature évènement : (Séminaire)
Evènement répétitif : (General ILL Seminar - College 8)
Site de l'évènement : Polygone scientifique

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