Photon Factory Activity Report 2017 #35 (2018)

BL-17A/2010G640, AR-NW12A/2013G654, BL-10C/2016R-56

X-ray Crystallography and SAXS Analysis of Calaxin, a Ca2+-Dependent Regulator ofFlagellar Motility
Takuya MIYAKAWA1, Yoshitaka MATSUMURA2, Kazuo INABA3, Masaki KOJIMA2 and Masaru
TANOKURA1,*
1
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2
Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0355, Japan
3Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan
1 Introduction
In many species, the female gamete or its associatedstructures release chemoattractants to attract spermatozoa.
The chemoattractant increases the intracellular Ca2+concentration of sperm, which is required for thedirectional changes of the sperm flagellum toward the egg.
An axonemal Ca2+-binding protein from ascidian Ciona
intestinalis, named calaxin, regulates the propagation ofCa2+-induced asymmetric flagellar bending throughbinding to a heavy chain (HC) of outer-arm dynein andtubulin in a Ca2+-dependent manner [1]. To elucidate therelationship between the Ca2+-dependent regulatory
mechanism and structural features of calaxin, we solved
crystal structures of calaxin in the Ca2+-bound (active)
and Mg2+-bound (inactive) forms. In addition, small-angle
X-ray scattering (SAXS) was performed to obtain theevidence of structural transition in solution.
2 Experiments
Calaxin was crystallized in the presence of Ca2+ orMg2+. For crystallographic phasing, the crystal wassoaked in a solution containing SmCl3, and the single
anomalous diffraction (SAD) data were collected at theSm peak wavelength of 1.600 Å on the BL-17A beamline.
The X-ray diffraction data of the Mg2+-bound calaxin
were collected at a wavelength of 1.000 Å on the ARNW12A beamline. All data were processed with the XDS
program to determine the crystal structures of calaxin.
The SAXS experiments were performed on the BL-10Cbeamline. All data were collected at a wavelength of1.488 Å and processed with the FIT2D program.
3 Results and Discussion
We determined the crystal structures of calaxin in theCa2+- and Mg2+-bound forms at 1.85 and 2.64 Åresolutions, respectively [2]. Calaxin was composed of 11helices and contained three EF-hand motifs that boundCa2+. The Ca2+-bound structure adopts the different
conformations between two calaxin molecules in an
asymmetric unit: One molecule is in the open state andthe other is in the closed state (Fig. 1). Compared with theopen state, the closed state exhibited the inward
movement of the helices α7, α8 and α10 and reduced theexposure of the hydrophobic surface. The crystal
structures are similar between the Ca2+- and Mg2+-boundforms (RMSD 0.516 Å), and the open and closed states
were also observed in the Mg2+-bound form. In the openstate, calaxin bound Mg2+ at the three EF-hand motifs.

However, no electron density of Mg2+ was observed inthird EF hand motif (EF3) of the closed state, and the side
chains of the loop between E-helix and F-helix seem to beflexible because of poor electron density. These structural
findings indicate that the affinity of EF3 for Mg2+ isdifferent between the open and closed states: Mg2+ prefers
to bind to the open state over the closed state.
To estimate the population of open and closed
conformers under equilibrium in solution, we performed
SAX experiments and fit the intrinsic scattering curves,which were theoretically calculated with crystal structuresof the two states of calaxin, to experimental scattering
curves [2]. Ca2+-bound calaxin adopts the closed andopen sates with abundance ratio of 18% and 82%,
respectively (Fig. 1). In contrast, the existing rate of
Mg2+-bound calaxin was limited to only open state(100%). These results suggest that the Ca 2+ binding
functions to shift open-closed structural transition toward
the closed state. In addition, the open state may bestabilized by a selective binding of Mg2+ to EF3 in theabsence of Ca2+.

Fig. 1: Open-closed structural transition of calaxin
References[1] K. Mizuno et al., Proc. Natl. Acad. Sci. U. S. A. 109,20497-20502 (2012).
[2] T. Shojima et al., Sci. Rep. 8, 2014 (2018).
* amtanok@mail.ecc.u-tokyo.ac.jp