Photon Factory Activity Report 2018 #36 (2019)

BL-17A, 1A/ 2018G523, 2016G559

Structure determinations of kinesin spindle protein Eg5 in complex with its inhibitors
1Hideshi Yokoyama1,*
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba,278-8510, Japan

1 Introduction
Kinesin spindle protein Eg5 is a mitotic spindle motor
protein, and plays an essential role in centrosome
separation and bipolar mitotic spindle formation during theearly stage of mitosis. The inhibition of Eg5 has beenshown to cause cell cycle arrest in mitosis with theirregular formation of monopolar spindles and subsequent
apoptotic cell death. As the inhibition of mitotic spindles
is a pharmaceutically validated strategy for cancer
therapeutics, human Eg5 represents an attractive target
molecule for novel clinical therapies. A large number of
Eg5 inhibitors have been reported. Some of them, such as
ispinesib and filanesib (ARRY-520), have been enteredinto clinical trials as anticancer drugs.
Our group has been previously determined the crystal
structure of the Eg5 motor domain in complex with abiphenyl-type inhibitor [1]. The inhibitor binds to the4/6 allosteric pocket 15 Å from the ATP-binding pocket,
which differs from conventional allosteric inhibitors thatbind to the allosteric L5/2/3 pocket of Eg5. Binding of
the inhibitor to the allosteric pocket induces thedeformation of the ATP-binding pocket through theTyr104 residue. The biphenyl-type inhibitor suppresses
the binding of ATP. The allosteric effect of the biphenyltype inhibitor on the binding of ATP is unique andunprecedented to the best of our knowledge.
Our group has been recently determined the crystal
structures of the Eg5 motor domain in complex with Strityl-L-cysteine (STLC)-type inhibitors [2].
2 Experiment
The Eg5 motor domain was expressed in Escherichia
coli BL21(DE3) CodonPlus RIL as a C-terminal His6
fusion protein using the expression plasmid pCold III.
Harvested cells were disrupted by sonication, and thecentrifuged supernatant was pufified with a Ni-NTA
Agarose resin. And then the eluted proteins were also
purified with a HiTrap SP HP cation-exchange column(GE Healthcare). The purified protein was mixed witheach inhibitor at a molar ratio of 1 : 5.
Crystallization was performed using the sitting-drop
vapor diffusion method at 20C. Crystallization drops
were prepared by mixing 0.5 L of the protein-inhibitor
solution and 0.5 L of the reservoir solution. In the caseof one protein-inhibitor complex, the protein-inhibitor
solution contained 17.4 mg/mL Eg5 motor domain, and thereservoir solution contained 30% (w/v) polyethylene
glycol (PEG) 3350, 0.1 M 2-morpholinoethanesulfonicacid (MES)-NaOH (pH 6.5), and 0.2 M ammonium sulfate.
Rod-shaped crystals grew to an approximate size of 0.1 0.05  0.05 mm.

A crystal was cryoprotected in a solution containing
30% (w/v) sucrose, and flash-frozen at 100 K. X-ray
diffraction data were collected, and data were processedand scaled with XDS and SCALA.
The structures were determined using a molecularreplacement method with the program MOLREP in theCCP4 suite. The structure of the Eg5 motor domain incomplex with STLC (PDB code, 2WOG) was used as an
initial model. Structural refinement was performed withREFMAC5 and PHENIX, and manual model fitting wasachieved with Coot.
3 Results and Discussion
The structures were determined at a resolution of 2.6 Åand 2.2 Å. All these structures contain two molecules inan asymmetric unit. The final model contains residues 16‒
366, one Mg2+ ADP, and one inhibitor for each molecules.
The structures of the Eg5 motor domain in complex with
STLC-type inhibitors were superposed well.
One inhibitor (here called as A) possesses a single paramethoxy substituent in one phenyl ring of the trityl groupin STLC. By superposing the structures in complex withthese inhibitors of STLC and inhibitor A, the structures ofthe binding site are nearly the same. Due to the presenceof the para-methoxy substituent of inhibitor A, however,
the C atom in the center of the trityl group of inhibitor A
was displaced 0.5 Å from that of STLC. For reference, thelocations of ADP are nearly identical in the STLC-typeinhibitor complexes.
Because the para-methoxy
substituent causes a steric hindrance with the neighboring
Right-side residues, the inhibitor molecule is totally shiftedby 0.5 Å to the Left-side wall when compared with STLC.
These differences may contribute to the higher affinity ofinhibitor A with Eg5 than that of STLC.
Crystallization and crystallographic analysis of anotherkinesin spindle protein CENP-E in complex with its
inhibitors are in progress.
Acknowledgement
This is the collaborative work with Prof. Akira Asai andhis lab members (Graduate School of PharmaceuticalSciences, University of Shizuoka). We thank the Photon
Factory staffs for assistance in X-ray diffraction
experiments.
References[1] H. Yokoyama et al., ACS Chem. Biol. 10, 1128 (2015)[2] H. Yokoyama et al., ACS Omega 3, 12284 (2018).
* yokoyama@rs.tus.ac.jp