Photon Factory Activity Report 2016 #34 (2017)

BL-1A, NW12A/2012G059, 2014G026

Crystal Structure of KIF19A Motor Domain with ADP
Doudou Wang1, Ryo Nitta2 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),
King Abdulaziz University, Jeddah 21589, Saudi Arabia
1

1 Introduction
The kinesin-8 motor, KIF19A, accumulates at cilia tips
and controls cilium length. Kif19a-/- mice displayedhydrocephalus and female infertility phenotypes due toabnormally elongated cilia that cannot generate proper
fluid flow[1]. Uniquely among kinesins, KIF19A
possesses the dual functions of motility along ciliary
microtubules and depolymerization of microtubules.
However, the molecular mechanisms of the dualfunctions of kinesin-8 proteins remain to be determined.
To elucidate the molecular mechanisms of thesefunctions we solved the crystal structure of its motor
domain and determined its cryo-electron microscopy
structure complexed with a microtubule. The features ofKIF19A that enable its dual function are clustered on its
microtubule-binding side [2].
In this report, we describe the details about the crystal
structure of KIF19A motor domain with ADP.
2 Experiment
The coding sequence of the mouse KIF19A motordomain (1-353, referred as KIF19AMD) was cloned into
pET21b (+) with a 7XHis-tag at the C-terminal of themotor domain. The KIF19A motor domain was purified
immobilized-metal affinity chromatography and ion
exchange chromatography and dialyzed against
crystallization buffer (10mM MOPS, pH 7.0, 100mM
NaCl, 1mM MgCl2, 0.1mM ADP, 15% Sucrose). The
hanging drop vapor diffusion method was used. One
microliter of KIF19A motor protein sample at 10 mg/ml
containing 0.1 mM ADP was mixed with 1 µl reservoir
buffer and incubated at 20°C. Single crystals are grownin 10% ethylene glycol, 2% PEG8000, 50 mM TrisBicine (pH 8.5), 9 mM MgCl2 and 9 mM CaCl2. The
crystals were frozen in liquid nitrogen within two weeks
after the crystal grew up. X-ray diffraction data werecollected using X-ray diffraction data were collectedusing a BL41XU beam-line (SPring-8, Japan), at awavelength λ=1.0 Å. The anomalous diffraction data
were collected using a BL-1A beamline (PhotonFactory, Japan), at the wavelength λ=2.7 Å. The
HKL2000 program package was used to index,integrate and scale the data. The structure of KIF19A
motor domain was solved
bymolecularreplacement
using
the Crystallography & NMR
System (CNS) and the atomic coordinates ofKIF18AMD (PDB: 3LRE)[3] as a search model.
Several rounds of iterative model building andrefinement were performed using COOT and Phenix. The

final crystallographic model of KIF19A motor domain
was refined to Rwork/Rfree of 0.222/0.302.
3 Results and Discussion
At the initial stage of the KIF19AMD structure
determination process, the residues of the switch II helix
α4 could not be determined because there was nocorresponding density at the site where α4 is usually
located at the center of the MT-binding interface. Instead,
we found an unmodeled helical-like density, which wasmore distant from the KIF19A catalytic core (Figure 1).
Long wavelength X-ray diffraction experiment was thus
performed to investigate the property of this density,
because in the switch II helix α4, one cysteine residue
exists. It successfully depicted the anomalous diffractionsof sulfur or phosphorus atoms. Among them, one strong
anomalous signal was detected close to the center of thecorresponding helical density. When the Cys283 residue
was assigned to this anomalous signal (Figure 2), all theresidues in α4 and the following loop L12 werereasonably determined.
The overall structure of the KIF19AMD motor domainshared a similar triangle-shape with other kinesins (Figure3). In the KIF19AMD atomic structure, Mg2+-ADP wasfound embedded in the nucleotide-binding pocket. Incomparison with previously solved motor domains ofvarious KIFs, the remarkable features of the KIF19A
motor domain are concentrated on its MT-binding sideand include the long and wide L2 loop, the flexible L8loop, the disordered α4 helix, and the short α6 helix(Figure 3).
The destabilized switch II helices contribute to theadaptation to the two distinct interfaces of straight andcurved MTs. The rotatable L8-α3-L9 cluster also
supports adaptation to the two interfaces. The basiccluster of L2, as well as the basic residues of L12, enables
KIF19A to tether to both straight and curved MTs via
flexible ionic interactions with the acidic residues of H12or the E-hook of tubulins. This assures its motility along
the MT, although motility speed may be decreased. The
hydrophobic tip of L2, as well as the surrounding basicand acidic clusters, plays critical roles in MT
depolymerization. These residues stabilize the intertubulin-dimer interface of the curved MT protofilament.
Thus, the curved conformation of MT ends is stabilizedby L2, resulting in the depolymerization of the MTs. In
this way, KIF19A has acquired dual functions by

Photon Factory Activity Report 2016 #34 (2017)

introducing multiple strategies. The resulting KIF19A is aslow plus-end directed motor combined with mild MTdepolymerizing activity.

Acknowledgement
We are grateful to N. Matsugaki from PF for thetechnical support. We thank members of the Hirokawa
lab for assistance and discussions.
References[1] S. Niwa et al., Dev Cell 23, 1167 (2012).
[2] D. Wang et al., eLife 5:e18101 (2016).
[3] C. Peters et al., EMBO J 29, 3437(2010)* hirokawa@m.u-tokyo.ac.jp

Figure 1 The unmodeled helix

Figure 2 The anomalous signal found around theunmodeled helical density.

Figure 3 The overall structure of KIF19AMD