Photon Factory Activity Report 2014 #32 (2015) B

NW12A/2008G023

Crystal Structure of the KIF5C Motor Domain Without Any Nucleotide
Shigeyuki Inoue,1 Ryo Nitta1,2 and Nobutaka Hirokawa1,3,*
Department of Cell Biology and Anatomy, Graduate School of Medicine,
The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
2
RIKEN Center for Life Science Technologies, Tsurumi, Yokohama 230-0045, Japan
3
Center of Excellence in Genomic Medicine Research (CEGMR),
KAU, Jeddah 21589, Saudi Arabia
11 Introduction
The molecular motor kinesin moves along microtubules(MTs) using energy from ATP hydrolysis in an initial
step coupled with ADP release. In neurons, kinesin1/KIF5C preferentially binds to the GTP-state MTs overGDP-state MTs to selectively enter an axon among many
processes [1]; however, because the atomic structure of
nucleotide-free KIF5C is unavailable, its molecularmechanism remains unresolved. Here, the crystal
structure of nucleotide-free KIF5C and the cryo-electron
microscopic structure of nucleotide-free KIF5C
complexed with the GTP-state MT (GMPCPP-MT) arepresented. In silico docking of the crystal structure withthe cryo-EM structure revealed the mutual
conformational changes of KIF5C and GMPCPP-MT.
Nucleotide-free KIF5C complexes with GMPCPP-MT toacquire a new conformation that we termed the “rigorconformation [2].” This conformation of KIF5C not only
provides an important missing link in the structural
analysis of kinesin, but also elucidates the molecularmechanism of the preferential binding of KIF5C to theGTP-MT.
In this report, we describe the details about the crystal
structure of nucleotide-free KIF5C motor domain.
2 Experiment
The KIF5C motor domain purified by immobilized
metal affinity chromatography and cation exchange
chromatography was dialyzed against crystallizationbuffer (10 mM PIPES, pH 7.4, 10 mM HEPES, pH 7.4,
100 mM NaCl, 1 mM MgCl 2, 1 mM EGTA, 20 % (w/v)
sucrose and 100 µM ADP, pH 7.0). Crystallization was
done using the hanging-drop vapor diffusion at 20 °C.
The reservoir buffer consisted of 2 M ammonium sulfate,
100 mM sodium citric acid, pH 5.5 and 16% (v/v)
glycerol. Crystals appear within a week in samples.
Subsequently, the C-terminal tail peptide solution ofKIF5C (EAVRAKNMARRAHSAQIAKPIRPG) wasadded to the drop to release bound ADP, such that thefinal concentration of the tail peptide was 1–2 mM. A
few minutes after the addition of the tail peptide, thecrystals were frozen in liquid nitrogen. X-ray diffraction
experiments were performed at −178 °C on a NW12A
beam line at the PF-AR (KEK, Tsukuba, Japan). The
data were processed using the program HKL2000. The

structures were determined using molecular replacement
methods, with the structure of KIF5 in the ADP state(PDB ID: 2KIN) as the initial model. Subsequent roundsof model building and refinement were performed using
the program Coot and the program Refmac5. Finally, themodel of the nucleotide-free KIF5C motor domain wasrefined to R and R-free values of 23.7 and 29.9%,
respectively.
3 Results and Discussion
Currently, the atomic structure of any of the KIFs
without nucleotide has not been available, except for thatof Kin I kinesin [3]. We therefore set out to solve thecrystal structure of KIF5C motor domain without any
bound nucleotide (Fig.1).
The weakly bound ADP without Mg2+ was found in
the nucleotide-binding pocket. We thus soaked several
additives to the weakly-ADP-bound crystals to further
accelerate the release of ADP. In the presence of the Cterminal peptide, only a small round electron density wasfound in the pocket instead of ADP, although thecorresponding density for the peptide was not found in
the electron density map possibly because of thepeptide's high flexibility (Fig.2).
The crystal structure of the KIF5C motor domain
without nucleotide adopts the ATP-like conformation.
Especially, the conformations of switch II and the necklinker are very similar to ATP-like structures. On theother hand, the switch I conformation of this model isslightly different from those in the other ATP-likestructures. In this structure, helix 3 rotates so that theC-terminal region of 3 and the following L9 moveaway from the nucleotide-binding pocket (Fig.3). Thisrotation observed here breaks the Mg-stabilizer, thus,
Mg-ADP is destabilized, which opens the pocket tofacilitate the exchange of ADP with Mg-ATP [4] .
References[1] T. Nakata et al., J Cell Biol 194, 245 (2011).
[2] M. Morikawa et al., EMBO J 34, 1270 (2015).
[3] K. Shipley et al., EMBO J 23, 1422 (2004).
[4] R. Nitta et al., Nat Struct Mol Biol 15, 1067 (2008).
* hirokawa@m.u-tokyo.ac.jp

Photon Factory Activity Report 2014 #32 (2015) B

Fig.2

Fig.1 Overall structure of nucleotide-free KIF5C motor
domainFig.3 The rotation of helix 3 compared with other state(PDB ID:1BG2 as an ADP state and 4HNA as an ADPAlF4 state)Fig.2 Electron density map of the nucleotide-binding
pocket of nucleotide-free KIF5C