Photon Factory Activity Report 2018 #36 (2019)

BL-5A, AR-NW12A/2016G516

X-Ray diffraction experiments for drug target protein of human casein kinase II
Chie SHIBAZAKI, Shigeki ARAI, Rumi SHIMIZU, Motoyasu ADACHINational Institutes for Quantum and Radiological Science and Technology,
2-4 Shirakata Tokai-mura, Naka-gun, Ibaraki, 319-1106, Japan
1 Introduction
Casein kinase II (CKII) exhibits broad phosphorylation
reaction on various important regulatory proteins such as
survival factors in eukaryotic cells. Since the relationshipof CKII over-expression to carcinogenesis and cancer
metastasis has been reported, CKII is considered to be oneof the drug target proteins. Here, we aimed to obtain thestructural information with location of hydration watermolecules, and to elucidate the catalytic reaction of CKII
for development of effective inhibitors.
2 Experiment
All crystals were prepared in vapor diffusion hanging
drops by a micro seeding method. A reservoir solution
containing 0.1 M Tris–HCl (pH 8.5), 1.2 M ammonium
sulfate, 5% acetonitrile, and 2 mM DTT was mixed withthe protein solution, and equilibrated at 293K before
seeding. The obtained crystals were soaked into a cryoprotectant solution, composed of 0.1 M Tris–HCl (pH 8.5),
35% PEG MME 5000, 0.2 M ammonium sulfate and 10%(w/v) glycerol, and were mounted with a cryoloop andflash-cooled in a nitrogen-gas stream at 100 K. The
collected data were indexed, integrated and scaled using
DENZO and SCALEPACK programs. The structuralrefinement was performed by the program PHENIX.
3 Results and Discussion
We have found the hydrogen bond network going
through the protein molecule from catalytic residue ofAsp156 to bulk solvent at the opposite side by neutron
crystallography [1]. Asp156 is well-known to enhance thenucleophilicity of the substrate OH group to the γ-phospho
group of ATP by elimination of the proton. Interestingly,
the network originating at Asp156 involves the ion pair of
His148 and Asp214 inside the protein molecule.
According to the neutron structure analysis, the hydrogen
atom in the water molecule bound to Asp214 displayed
delocalization in a distance of 0.2 Å probably because ofquantum effect. Additionally, we confirmed the catalytic
activity of CKII and mutant enzymes. The results indicatedthat the mutations on His148 to Ala, Ser, and Asn
significantly affect to the catalytic activity. Table 1 lists thedata collection and refinement statistics. The analysis
revealed water molecules occupying in the space of side
chain in the all mutants, and can keep hydrogen bonding[1]. An example in H148A is shown in Fig. 1. These results
demonstrate that the ion pair of His148 and Asp214 plays
a key role in catalytic reaction at the active site, and thelong and potential hydrogen bonding network may assist
the general base catalyst to extract a proton with link to thebulk solvent via intermediates of the ion pair. Thesefindings will lead us to know the catalytic mechanism moredeeply for drug design.

Table 1: Diffraction data and refinement statistics
Protein

WT1*1

H148A*1

H148S*1

H148N*1

Space Group*2

P21

P21

P21

P21

WT2
P21

Temperature

100

100

100

100

100

Resolution

1.05

1.20

1.50

1.65

1.70

No. of ref.*3

137,800

92,18949,69737,33834,389

Redundancy

6.4

4.5

4.7

3.3

3.3

R-merge

0.042

0.058

0.036

0.039

0.043
0.342

(outer shell)0.591

0.563

0.540

0.336

Completeness

0.945

0.955

0.999

0.998

0.999

(outer shell)

0.906

0.927

0.999

0.997

0.998

Refinement
R-factor

0.160

0.152

0.194

0.197

0.184

Free-R

0.180

0.181

0.230

0.239

0.222

PDBID

5ZN1

5ZN2

5ZN3

5ZN4

5ZN5

*1; two mutations of C147A and C220A are included.
*2; unit cell parameters for all derivatives are similar to that of WT1(a=58.6, b=45.5, c=63.2 Å, β=111.8˚).
*3; number of unique reflections.

Fig. 1: Refined X-ray crystal structures of WT1 (a) andH148A mutant (b). The two additional water molecules areobserved in the space near Ala148 in (b).

Acknowledgement
We greatly thank the PF staff for diffraction datacollection.
References[1] C. Shibazaki et al., J. Mol. Biol. 430, 5094-5104 (2018).
* adachi.motoyasu@qst.go.jp