Scientific and Educational Reports of the Facultyof Science and Technology, Kochi University
Vol. 2 (2019), No. 6

Crystallization and preliminary crystallographic studies of arginine
kinase from the ciliate Paramecium tetraurelia
Yumeto Otsuka†1, Daichi Yano†2, Junko Tanaka1, Kouji Uda2, Tomohiko Suzuki2, Shigeru Sugiyama2*
1 Graduate School of Science, Kochi University, Kochi 780-8520, Japan
2 Faculty of Science & Technology, Kochi University, Kochi 780-8520, Japan† These authors contributed equally to this work.
* Correspondence e-mail: ssugiyama@kochi-u.ac.jp
KEYWORDS. arginine kinase, protein crystallization, X-ray crystallography
ABSTRACT: The arginine kinase from the ciliate Paramecium tetraurelia catalyzes the reversible transfer of phosphoryl
groups from adenosine triphosphate to arginine, generating
adenosine diphosphate and arginine phosphate. The ciliate
Paramecium tetraurelia has four arginine kinases (PtAK1 to4). PtAK3 showed typical substrate inhibition toward arginine,
and the enzymatic activity markedly decreased when the arginine concentration increased. To elucidate the substrate inhibition mechanism of PtAK3, we performed a crystallographic
study of PtAK3. Here we report the crystallization and preliminary X-ray diffraction analysis of PtAK3. The diffraction data
were collected and processed with a 2.6 Å resolution based on
the PtAK3 crystals. The preliminary crystallographic analysis
revealed that the PtAK3 crystals belonged to the orthorhombic
space group P212121, with unit-cell parameters a = 77.5, b =162.5, c = 68.8 Å. They contained two molecules per asymmetric unit.

1. Introduction
Phosphagen kinases (PKs) are enzymes that catalyze the reversible transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to guanidine substrates, generating adenosine diphosphate (ADP) and phosphorylated guanidine compounds referred to as phosphagens (Morrison & James, 1965).
These enzymes play a key role in maintaining a constant ATP
concentration, especially in cells that consume a significant
amount of ATP energy (Ellinton, 2001, Ellington & Suzuki
2006). Arginine kinase (AK), a member of the phosphagen
kinase family, is widely distributed in invertebrates (Uda et al.,2006), including unicellular organisms such as bacteria (Andrews et al., 2008; Suzuki et al., 2013) and protozoan ciliates(Uda et al., 2006; Magida & Fraga, 2007). Substrate inhibitionis the phenomenon where enzymes are inhibited by their own
substrate. This occurs approximately for 20% of all enzymes(Chaplin & Bucke, 1990) and often has important biological
functions (Kuehl, 1994; Reed et al., 2010).
The ciliate Paramecium tetraurelia contains four types ofarginine kinase (PtAK1 to PtAK4). In a previous study (Yanoet al., 2017), we showed that PtAK3 is characterized by typical arginine substrate inhibition and that enzymatic activity
markedly decreased with a concentration of 1 mM arginine
substrate (Yano et al., 2017; Yano & Suzuki, 2018). Substrateinhibition in wild-type PKs was first observed with PtAK3. In
these same studies—by using three models—the substrateinhibition kinetics of wild-type and mutant PtAK3 were also
Received: May 5, 2019
Reviewed by anonymous referee(s), and accepted: May 31, 2019
Published: July 24, 2019analyzed to understand the various occurring mechanisms. The
results suggested that after primary arginine substrate binding,the binding of another arginine at the secondarily induced
inhibitory site is accelerated to form the two substrate-enzyme
complex, causing substrate inhibition (Yano & Suzuki, 2018).
In the current study, we conducted a crystallographic investigation to elucidate the reaction mechanism of PtAK3 at theatomic level.

2. Materials and Methods
2.1. Crystallization
Recombinant PtAK3 was purified according to the protocol
described previously (Yano et al., 2017). The resulting recombinant PtAK3 was dialyzed in 10 mM Tris-HCl (pH 8.0). For
crystallization, the purified PtAK3 solution was concentrated
to 18 mg/ml by the ultrafiltration method using an Amicon
Ultra-4 concentrator (Merck, USA). The purity was verified
by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) on 12.5% gels (Laemmli, 1970). A preliminary
search for crystallization conditions was performed using thesitting-drop method in 24-well Cryschem plates (HamptonResearch, USA). Initial crystallization screening conditions
were constructed using several commercially available kits:

Figure 1.
A 12.5% SDS-PAGE of purified PtAK3. The gel was stained with Coomassie Brilliant Blue. Lane M, molecular-mass markers (labelled in kDa);
Lane 1, purified PtAK3.

1

Crystal Screen and Crystal Screen 2 (both from Hampton Research, USA). Typically, a 1.0 μl droplet consisting of 18mg/ml PtAK3 in 10 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol was mixed with an equal volume of reservoir solution.
This droplet was allowed to equilibrate against 500 μl of reservoir solution. All crystallizations were carried out at 285 K.

was obtained with a reservoir solution consisting of 20% (w/v)
PEG4000 and 0.1 M Hepes-NaOH (pH 7.0). The crystallization condition is summarized in Table 1.
Table 1.
Crystallization conditions

2.2. X-ray data collection
Data collection was performed with the beamline BL26B1 ofthe SPring-8 synchrotron-radiation source (Hyogo, Japan).
The crystals were soaked in a cryoprotectant solution and thenmounted in a cryoloop. The crystal was immediately flashcooled in a stream of nitrogen gas at 100 K. Diffraction data
sets were collected using 0.5° oscillations with a crystal-todetector distance of 200 mm. The diffraction data were processed and scaled using the HKL-2000 program package (Otwinowski & Minor, 1997).

MethodPlate type
Temperature (K)Protein concentration (mg/ml)
Buffer composition of protein solution
Composition of reservoir solution
Volume and ratio of drop
Volume of reservoir (µl)

Sitting-drop vapour diffusion
24-well plate285
1010 mM Tris-HCl (pH 8.0),
1.0 mM DTT
0.1 M Hepes-NaOH (pH 7.0),
20% (w/v) PEG4000
2 μl, 1:1 (protein solution:reservoir solution)
900

Table 2.
Crystal parameters and data-collection statistics for PtAK3.
Values in parentheses are for the outer shell.

Figure 2.
Crystal of PtAK3 prepared by sitting-drop vapour diffusion.

X-ray source
Wavelength (Å)
Temperature (K)Detector
Crystal-to-detector distance (mm)Rotation range per image (°)
Total rotation range (°)
Exposure time per image (s)Space group
Unit-cell parametersa, b, c (Å)α, β, γ (°)
Mosaicity (°)
Resolution range (Å)(High-resolution shell)
Total No. of reflections
No. of unique reflections
Completeness (%)
Multiplicity
Mean I/σ(I)Rmerge§ (%)
Overall B factor from Wilson plot (Å2)

SPring-8 (BL26B1)
0.90
100
Eiger 4M CCD
200
0.5
211.5
20P212121
77.5, 162.5, 68.890, 90, 90
0.29
50 - 2.60(2.64- 2.60)
15002524813
88.4 (74.1)
6.0 (3.6)
4.4 (1.7)
18.6 (56.3)
24.8§Rmerge = ∑hkl∑i |Ii(hkl) ‒ ‹I(hkl)›|/∑hkl∑i Ii(hkl), where Ii(hkl) is the ith
observed intensity of reflection hkl and <I(hkl)> is the average intensity
over symmetry-equivalent measurements.

3. Results and Discussion
Purified PtAK3–with a calculated molecular weight of 44.6
kDa–migrated as a single band on the matrix gel using SDS–
PAGE. The analysis showed a homogeneous preparation witha molecular weight of 45 kDa (Fig. 1). A preliminary searchfor crystallization conditions was performed using Hampton
Crystal Screens. Microcrystals of PtAK3 were obtained asclusters from several PEG-based reagents. The crystals fromthe cluster exhibited poor diffraction quality and were too
small for diffraction experiments. The use of a seeding technique in an attempt to improve the crystals proved unsuccessful. Therefore, we tried to further refine the crystallization
conditions. Consequently, the crystallization conditions wereoptimized by changing the PEG concentration and the pH. Asa result, suitable PtAK3 crystals with dimensions of 0.02 ×0.02 × 0.2 mm appeared at 285 K within a week. The best
crystallization condition for X-ray diffraction measurement

X-ray diffraction experiments were performed under astream of nitrogen gas at 100 K. A crystal was mounted in anylon loop, soaked rapidly in the 15% (v/v) glycerol, 20%(w/v) PEG4000, 0.1 M Hepes-NaOH (pH 7.0) and then immediately flash-cooled. The wavelength was fixed at 0.9 Å. A
total of 423 frames were recorded with an oscillation angle of0.5°, an exposure time of 20 s per frame and a crystal–to–
detector distance of 200 mm. Diffraction data were processedand scaled using the HKL-2000 program package (Otwinowski & Minor, 1997). Analysis of the symmetry and the systematic absences in the recorded diffraction pattern indicated thatthe crystals belonged to the cubic space group P212121, withunit-cell parameters a = 77.5, b = 162.5, c = 68.8 Å. Assuming
the presence of two PtAK3 molecules (2 × 45 kDa) in theasymmetric unit, the VM value is calculated to be 3.1 Å3Da-1,with an estimated solvent content of 49.4 %; these values are2

within the range commonly observed for protein crystals (Matthews, 1968). Calculation of the Matthews coefficient suggested that two monomers exist in the asymmetric unit. A totalof 150025 observed reflections were merged into 24813
unique reflections in the 50.0-2.6 Å resolution range. Datacollection and processing statistics are summarized in Table 2.
Structural analysis of the PtAK3 is now in progress. We anticipate that our structural study of the PtAK3 will contribute toelucidation of the substrate inhibition mechanism of thePtAK3.

Acknowledgements
The authors are grateful to Seiki Baba, Nobuhiro Mizuno,
Akifumi Higashiura, Eiki Yamashita, and Atsushi Nakagawa
for their kind help in data collection at BL26B1 and BL44XUof SPring-8. The synchrotron-radiation experiments were performed at SPring-8 with the approval of the Japan Synchrotron
Radiation Research Institute (JASRI; Proposals 2016A/B6623,
2017B2516 2018A/B6824, and 2018A/B2509). This work wassupported in part by the Takahashi Industrial and Economic
Research Foundation and by JSPS KAKENHI grant Nos.
16K07270 and 19K06588 to SS.

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