Photon Factory Activity Report 2017 #35 (2018)

BL-15A2, BL-10C, BL-6A/2014R-48, 2015R-05, 2016R-21

SEC-SAXS Analysis Revealed the Transitional Complex ofMicrotubule Depolymerizing Kinesin and Tubulin in Solution
Tadayuki OGAWA1, Shinya SAIJO2, Nobutaka SHIMIZU2, and Nobutaka HIROKAWA1,*
1
Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo,
7-3-1 Hongo, Tokyo, 113-0033, Japan2
Photon Factory, 1-1 Oho, Tsukuba, 305-0801, Japan
1 Introduction
Microtubules (MTs) are dynamic tube-like structurespolymerized from large numbers of tubulin dimerproteins. The proper regulation of the polymerization anddepolymerization of MTs is critical for fundamental
events in cells. KIF2, a member of kinesin motor proteins(KIFs), is known to regulate MTs dynamics bydepolymerizing MTs from tip ends [1][2]; however, themechanism by which a small number of KIF2 proteins
drives the processive depolymerization of MTs composedof a large number of tubulins remained unclear. To
address this issue, the transitional states of MTs
depolymerization via KIF2 were analyzed by the smallangle X-ray scattering (SAXS), in combination with sizeexclusion chromatography (SEC) and multi-angle lightscattering (MALS) [3][4].
2 ExperimentDuring MTs depolymerization via KIF2, the largetransitional KIF2-tubulin complex was monitored in highresolution size-exclusion chromatography (HiRes SEC)[3]; therefore, the large complex was further analyzed bySAXS at beamline BL-15A2 [5]. HiRes SEC wasconnected with the following flow-cell for the SAXS
measurement, and the separated sample was directlyanalyzed by SAXS (HiRes SEC-SAXS), by which the Xray damage to the protein complex was greatly reduced.
In addition, HiRes SEC-RI-MALS was also conducted toconfirm the molar mass value of the complex. In HiRes
SEC-SAXS system, the mono-disperse eluate directly
flows into a flow-cell holder and serial scattering images
were taken with 10 sec exposure. Each ten scatteringprofiles in the first half of an elution peak were averaged
and the scattering profiles of the KIF2-tubulin complex
extrapolated to an infinite dilution condition werecalculated from these averaged scattering profiles.
3 Results and Discussion
The radius of gyration (Rg) was estimated from theGuinier approximation to be 51.5±0.5 Å, and Dmax wascalculated to be 203 Å on the basis of the pair distributionfunction, P(r). Both HiRes SEC-RI-MALS and HiRes
SEC-SAXS estimated the large transitional KIF2-tubulin
complex around 250 kDa in molecular weight, whichcorresponded the sum of its components: 51 kDa (KIF2
core domain) and two sets of 103 kDa (tubulin dimer)(1:2 complex). These measurements directly suggested
that one KIF2 forms a large complex with two sets oftubulin dimers as a transitional conformation. To reveal
the structural arrangement and outline of the KIF2-tubulin
complex in solution, HiRes SEC-SAXS data was further

analyzed, and SAXS model of the large 1:2 complex
displayed a pipe structure with a bulge at one end and hada reasonable volume for its components, one KIF2 andtwo tubulin dimers, arranged longitudinally (Figure 1).
In an in vivo system, large amounts of MTs must be
regulated efficiently by using the limited resources ofKIF2 proteins and ATP. In this sense, the longitudinal 1:2
complex is a fundamental property of the catalytic
mechanism of MTs depolymerization. Therefore, KIF2
functions as a smart molecular machine that efficiently
depolymerizes two sets of tubulin dimers with one ATP.
The microtubule depolymerization mechanism revealedin this study promises to become the basis forunderstanding the pathology to establish effective
treatment methods for microtubule-related diseases.

Fig 1: Structure of KIF2-Tubulin Complex in Solution
Acknowledgement
This work was supported by the Platform for Drug
Discovery, Informatics, and Structural Life Science(PDIS) (Proposal No: 2048). We thank the all PF staff for
great assistance.
References[1] T. Ogawa et al., Cell 116(4):591 (2004).
[2] T. Ogawa, N. Hirokawa, Cell Rep. 12(11):1774(2015).
[3] T. Ogawa et al., Cell Rep. 20(11):2626 (2017)[4] T. Ogawa, N. Hirokawa, Biophys Rev. 10(2):299-306(2018)[5] P. Bernadó, N. Shimizu, et al., Biochim Biophys Acta.
1862(2):253 (2018)* hirokawa@m.u-tokyo.ac.jp