Photon Factory Activity Report 2014 #32 (2015) B

AR-NW12A/2013G126

Crystallographic analysis of cytokine BMP-10
Nobutaka Numoto*
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
Bunkyo-ku, Tokyo 113-8510, Japan
1 Introduction
Bone morphogenetic protein 10 (BMP-10) is a memberof the TGF-β superfamily and plays a critical role in heartdevelopment. A substitution variant of BMP-10 gene has
been found to be associated with hypertensive dilated
cardiomyopathy. [1] BMP-10 is translated as a precursor
protein with an N-terminal propeptide and a C-terminal
region (active BMP-10) that is cleaved by a protease suchas furin. [2] The mature protein consists of both thepropeptide and BMP-10 as a complex, and interactions of
this mature protein with the target receptors would
promote releasing the activated BMP-10. To elucidate thedetailed molecular mechanisms of maturation, activation,and pathogenesis of BMP-10, we have initiatedcrystallographic studies for human BMP-10. In this study,
we report the preliminary X-ray diffraction analysis ofhuman BMP-10.
2 Experiment
Human BMP-10 were expressed in HEK293-EBNAcells and purified from the culture supernatants. Initial
crystallization conditions were screened by the sparse
matrix method [3] using commercially available
screening kits (Hampton Research) with the hanging-drop
vapor diffusion method. Heavy atom derivative crystals
were prepared by soaking in reservoir solutions
containing 1-10 mM HgCl2, Methylmercury(II) chloride,or K2PtCl4. Prior to X-ray diffraction experiments, thecrystals were soaked in the mother liquor containing 2025% glycerol as a cryoprotectant and flash-frozen in anitrogen gas stream at 95 K. X-ray diffraction
experiments were performed at beameline NW12A at PFAR, KEK. The data were processed and scaled using theHKL2000 package [4] and were truncated using theCCP4 program suite. [5] Self-rotation function was
calculated using MOLREP. [6]3 Results and Discussion
The crystals of BMP-10 (Fig. 1) appeared within 7-14
days with typical dimensions of 0.2 × 0.2 × 0.2 mm3. For
the best set, the crystal diffracted X-rays up to 3.6 Åresolution. The detector, camera distance, wavelength,
oscillation range, and exposure time were ADSC Q210r,279.4 mm, 1.0000 Å, 0.5 degree, and 20.0 sec without an
attenuator, respectively. A total of 180 frames wereobtained.
The crystal belongs to the space group I222 or I212121,
with unit-cell parameters a = 183.0, b = 184.6, c = 186.0
Å. Assuming that 3 or 6 molecules are contained in an
asymmetric unit, the Matthews coefficient VM [7] iscalculated to be 5.5, 2.7 Å3/Da, corresponding to 77.5,
55.0% solvent content, respectively. Self-rotation
function clearly show that BMP-10 molecules have noncrystallographic 2, 3, and 4 fold axes. These facts suggestthat BMP-10 forms 12 or 24-meric oligomer with anoctahedral pseudo-symmetry.
Since no clear solutions were obtained by themolecular replacement (MR) method, the heavy atom
derivatives were prepared for the multiple isomorphs
replacement (MIR) method. We have collected the datafrom the HgCl2 derivatives at 4.2 and 5.0 Å resolution,and K2PtCl4 derivatives at 6.5 Å resolution. Due to thesevere non-isomorphism of these derivative crystals, nointerpretable peaks were observed in the difference
Patterson maps. Combination of the MR and MIR
methods is in progress.

Fig. 1: Crystals of BMP-10.
References[1] N. Nakano et al., Am. J. Physiol. Heart Circ. Physiol.
293, H3396 (2007).
[2] D. Susan-Resiga et al., J. Biol. Chem. 286, 22785(2011).
[3] J. Jancarik et al., J. Appl. Crystallogr. 24, 409 (1991).
[4] Z. Otwinowski and W. Minor, Methods Enzymol. 276,307 (1997).
[5] M. D. Winn et al., Acta Cryst. D67, 235 (2011).
[6] A. Vagin, and A. Teplyakov, Acta Cryst. D66, 22(2010).
[7] B.W. Matthews, J. Mol. Biol. 33, 491 (1968).
* numoto.str@mri.tmd.ac.jp