Photon Factory Activity Report 2022 #40 (2023)

BL-17A/2022G595, 2020G531

Structure of kinesin CENP-E motor domain in complex with an ATP analogue
Hideshi YOKOYAMA1,*, Akira SUZUKI1, Asuka SHIBUYA2

1 Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda,

Chiba, 278-8510, Japan

2 Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane City,

Chiba 283-8555, Japan

1 Introduction
Centromere-associated protein E (CENP-E) is akinesin motor protein essential for mitosis and a newtarget for anticancer agents with less side effects. To
rationally design anticancer drug candidates basedon structure, it is important to determine the threedimensional structure of the CENP-E motor domainbound to its inhibitor.
No CENP-E inhibitors have been approved for use
in clinical practice. To efficiently develop CENP-E
inhibitors with different chemical structures, it isuseful to have structural information on various typesof the CENP-E motor domain. However, only two
crystal structures of the CENP-E motor domain havebeen reported, both of which are in ADP-bound form
(PDB ID: 1T5C, 6M4I) [1], [2]. There are no crystalstructures of the CENP-E motor domain in complex
with its inhibitors.
We attempted to obtain the structural informationof the CENP-E motor domain bound to ATPcompetitive inhibitors by using an ADP hydrolysis
enzyme apyrase to replace ADP.
Here wedetermined the first crystal structure of the CENP-Emotor domain in complex with adenylylimidodiphosphate (AMPPNP), a non-hydrolysable
ATP analogue [3].
2 Experiment
The CENP-E motor domain was expressed andpurified as described previously [2]. The purified
protein was mixed with apyrase and AMPPNP at amolar ratio of 1 : 10 : 5, and incubated in 10 mM MESNaOH (pH 6.0), 4 mM EDTA, and 6 mM CaCl2 at
25 °C for two hours in order to remove ADP from thenucleotide-binding pocket of the CENP-E motor
domain. The resulting protein was purified by gel
filtration chromatography.
Crystallization wasperformed using the sitting-drop vapor-diffusion
method at 4 °C. The 8.3 mg/mL protein solution wasmixed with AMPPNP at a molar ratio of 1 : 50.
Crystallization drops were prepared by mixing theCENP-E motor domain and AMPPNP solution
described above, and the reservoir solution
containing 0.1 M MES-NaOH (pH 6.5) and 18% (w/v)
PEG3350. Needle-shaped crystals appeared after
five days.
A crystal was cryoprotected in a solution containing
25 mM PIPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM

MgCl2, 0.5 mM TCEP, 0.5 mM EGTA, 9% (w/v)
PEG3350, 500 mM MES (pH 6.5), 2.5% (w/v)
sucrose, 5.20 mM AMPPNP and 37.5% (w/v) glycerol,and ﬂash-cooled at 95 K.
X-ray diﬀraction data were collected and processedand scaled with the program XDS and SCALA. The
structure was determined by the molecularreplacement method with the program MOLREP inthe CCP4 suite. The structure of CENP-E-MgADP(PDB ID: 6M4I) was used as an initial model.
Structural reﬁnement was performed with REFMAC5.
Manual model ﬁtting was achieved with COOT.
3 Results and Discussion
The structure of CENP-E motor domain in complex
with AMPPNP was successfully determined at aresolution of 1.8 Å, which is hig her resolution than thepreviously reported structures in ADP-bound form:
1.9 Å resolution [2] and 2.5 Å resolution [1]. The
electron density of AMPPNP was observed in thenucleotide-binding site.
Structural differences between the structures ofCENP-E-AMPPNP and CENP-E-ADP are observed
near the nucleotide-binding site. The helix α4 of
CENP-E-AMPPNP was longer than CENP-E-MgADP.
The structure of CENP-E-AMPPNP reported in thisstudy was compared with the previously determined
structures of some kinesins in complex with AMPPNP.
The loop L5 of CENP-E-AMPPNP is shorter thanother kinesins, as CENP-E has fewer residues at loop
L5. We think that loop L5 is involved with the featureof CENP-E that the release of ADP is a rate-limiting
step in the ATPase cycle.
The methods of preparation and crystallization inthis study will be useful for crystallization of CENP-E
in complex with its inhibitors. Crystallization andcrystallographic analysis of CENP-E motor domain incomplex with its inhibitors are in progress.
Acknowledgement
This is the collaborative work with Prof. Akira Asaiand his lab members (Graduate School ofPharmaceutical Sciences, University of Shizuoka).
We thank the Photon Factory staffs for assistance in
X-ray diffraction experiments.
References[1] I. Garcia-Saez et al., J Mol Biol. 340, 1107 (2004).

Photon Factory Activity Report 2022 #40 (2023)[2] A. Shibuya et al., Acta Crystallogr. D 77, 280(2021).
[3] A. Shibuya et al., FEBS Lett. 597, 1138 (2023).
* yokoyama@rs.tus.ac.jp