research communications
Crystallization and X-ray
of the CH domain of the cotton kinesin GhKCH2aCollege of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People's Republic of China, and bSchool of Aerospace Medicine, The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi'an 710032, People's Republic of China
*Correspondence e-mail: liu@cau.edu.cn
GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to P21, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the with a Matthews coefficient of 2.22 Å3 Da−1, corresponding to a solvent content of 44.8%.
Keywords: plant-specific kinesin; CH domain; cotton kinesin GhKCH2.
1. Introduction
Microfilaments and microtubules, two vital cytoskeleton systems in cells, together take part in a variety of cellular activities, such as cell division and proliferation, transportation of ; Petrášek & Schwarzerová, 2009). The kinesins are a superfamily of microtubule-based motor proteins (Howard, 1996), some of which (KCHs) contain a single N-terminal calponin homology (CH) domain that is able to bind to both microfilaments and microtubules. In 2004, the first KCH (GhKCH1) was identified and demonstrated to be involved in the elongation of cotton fibres (Preuss et al., 2004). Subsequently, our laboratory and others identified further KCHs (GhKCH2, O12/OsKCH1, AtKinG and NtKCH1), all of which were able to bind to both microfilaments and microtubules in vitro or in vivo (Frey et al., 2009; Xu et al., 2009; Buschmann et al., 2011; Umezu et al., 2011; Klotz & Nick, 2012). Recently, Ram Dixit deduced that KCH might be involved in the transportation of actin fragments (Dixit, 2012).
and vesicles, and the organization and formation of plant preprophase bands, phragmoplasts and cell plates (Wasteneys & Galway, 2003The actin-binding CH domain, named after its first identification in calponin, consists of about 100 amino-acid residues (Takahashi & Nadal-Ginard, 1991). Since the first of the CH domain was published in 1997 (spectrin from Homo sapiens; PDB entry 1aa2 ; Djinovic Carugo et al., 1997), along with the first NMR structure published in 2002 (calponin from Gallus gallus; PDB entry 1h67 ; Bramham et al., 2002), increasing numbers of structures of CH domains from yeast, animals and humans have been solved and deposited in the PDB (https://www.rcsb.org ). In comparison, knowledge of plant CH-domain crystal structures remains limited, with only one structure available (fimbrin from Arabidopsis thaliana; PDB entry 1pxy ; Klein et al., 2004).
GhKCH2 (GenBank accession No. EF432568), a KCH previously cloned from cotton (Gossypium hirsutum) fibres in our laboratory, has a motor domain with microtubule-stimulated ATPase activity and a CH domain that strongly interacts with microfilaments, suggesting it to be a candidate for a linker between microfilaments and microtubules (Xu et al., 2007, 2009). The CH domain of GhKCH2 shares 31% amino-acid sequence identity with human calponin 1 and 28% amino-acid sequence identity with Arabidopsis fimbrin. Our previous studies revealed that GhKCH2-N (amino acids 1–306), containing the CH domain, had a higher affinity for F-actin (Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins (Kd = ∼5–50 µM) (Gimona et al., 2002; Xu et al., 2009). To elucidate the mechanisms of this unique biochemical feature, further exploration of the structure of GhKCH2-CH was performed. Here, the expression, purification, crystallization and preliminary X-ray diffraction studies of the CH domain of GhKCH2 are described.
2. Materials and methods
2.1. Protein expression and purification
The CH domain of GhKCH2 was cloned using the forward primer 5′-GAG AGT CCA TAT GGA TTT GGA ATC TAG AAA AGC TG-3′ (NdeI site in bold) and the reverse primer 5′-CAA CTC GAG TTA CGA GAG CCT CCA CTC GTT ATA G-3′ (XhoI site in bold). The PCR product was digested and inserted into a modified pGEX-4T-2 vector (kindly provided by Professor Zhongzhou Chen, China Agricultural University) at the NdeI and XhoI restriction sites with a TEV protease cleavage site between GST and the target gene (Table 1). The correct certified constructs were transformed into Escherichia coli strain BL21(DE3). Cells were grown at 37°C in LB medium containing 100 mg ml−1 ampicillin to an A600 of 0.6–0.8 and were induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 22°C overnight. The cells were harvested by centrifugation and lysed by gentle sonication in lysis buffer (0.1 M Tris–HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM PMSF). After high-speed centrifugation, the supernatant was loaded onto a home-made Glutathione Sepharose 4B column (GE Healthcare) and incubated for 1 h. After washing with wash buffer (0.1 M Tris–HCl pH 7.5, 150 mM NaCl), TEV protease was added to cleave the fusion protein overnight. The collected flowthrough was purified by Mono Q and further polished by gel filtration on a HiLoad 16/60 Superdex 75 pg column (GE Healthcare). The purified proteins were pooled, concentrated to 10–15 mg ml−1, flash-cooled in liquid nitrogen and stored at 80°C. Protein purity and identity were assessed by SDS–PAGE with Coomassie Bright Blue staining (Fig. 1). All of the purification procedures described above were conducted at 4°C.
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2.2. Protein crystallization
Initial screening for crystallization conditions was carried out in 48-well sitting-drop plates using the commercially available kits Crystal Screen, Crystal Screen 2 and Index (Hampton Research, USA) at 4°C. Crystals of GhKCH2-CH were initially grown from a mixture of 1 µl protein solution (10–15 mg ml−1 in 20 mM Tris–HCl pH 7.5, 150 mM NaCl) and 1 µl precipitatant solution equilibrated against 100 µl reservoir solution at 277 K.
Subsequent optimizations were performed using 24-well sitting-drop plates, and the size of the crystals was enlarged by streak-seeding using a cat whisker (Figs. 2a and 2b). Detailed information on GhKCH2-CH crystallization is given in Table 2.
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2.3. Data collection
Mounted crystals were dehydrated with a solution consisting of 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000, 10%(v/v) DMSO for 5 min, transferred to a solution consisting of 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000, 20%(v/v) DMSO for a further 5 min and finally flash-cooled in liquid nitrogen. The diffraction data set was collected at 100 K on BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Q315 CCD. A total of 360 images with an oscillation angle of 1° each were collected using a 250 mm crystal-to-detector distance and an exposure time of 1 s per frame (Fig. 3). Detailed information on data collection is given in Table 3.
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3. Results and discussion
Indexing with XDS (Kabsch, 2010) indicated that GhKCH2-CH crystallized in P21. Analysis of the with phenix.xtriage revealed a significant off-origin peak that was 78.6% of the height of the origin peak (Adams et al., 2010). Analysis of average intensities with TRUNCATE showed that the crystals had strong reflections with indices of l = 2n [even reflections, I/σ(I) = 23.6] and weak reflections with indices of l = 2n + 1 [odd reflections, I/σ(I) = 13.3] (French & Wilson, 1978). HHpred (https://toolkit.tuebingen.mpg.de/hhpred ; Hildebrand et al., 2009) was used to identify homologues of the GhKCH2 CH domain, and the ten PDB hits (PDB entries 1p2x , 1ujo , 1h67 , 1p5s , 1wym , 3i6x , 1wyr , 213g , 1wyp and 1wyn ) with the highest scores were used as search models. When determining the of GhKCH2-CH using Phaser (McCoy et al., 2007), the structure of human calponin 2 (PDB entry 1wyn ; RIKEN Structural Genomics/Proteomics Initiative, unpublished work) gave the best results after further among these models. Four molecules were found in the with a Matthews coefficient of 2.22 Å3 Da−1, corresponding to a solvent content of 44.8%.
The entire reflection data set was split into odd (l = 2n + 1) and even components (l = 2n) using MTZUTILS from the CCP4 package (Winn et al., 2011). The positions of the four molecules in the were optimized using rigid-body with odd reflections, and was subsequently performed using REFMAC or phenix.refine with even reflections (Murshudov et al., 2011; Oksanen et al., 2006; Adams et al., 2010). After several cycles of such the model was refined against all data to an Rfree of about 35%. Owing to the low amino-acid sequence identity of the GhKCH2 CH domain to other CH domain-containing proteins, crystallization of selenomethionine-labelled protein is in progress.
Acknowledgements
We thank the staff of beamline BL17U1 at SSRF for their assistance during data collection. This work was supported by an Award of Outstanding PhD Dissertation from Beijing Municipal Commission of Education (Project 20111001903), the China Postdoctoral Science Foundation (Grant No. 2014M562597) and the National Natural Science Foundation of China (Grant No. 31500928).
References
Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. Web of Science CrossRef CAS IUCr Journals Google Scholar
Bramham, J., Hodgkinson, J. L., Smith, B. O., Uhrín, D., Barlow, P. N. & Winder, S. J. (2002). Structure, 10, 249–258. CrossRef PubMed CAS Google Scholar
Buschmann, H., Green, P., Sambade, A., Doonan, J. H. & Lloyd, C. W. (2011). New Phytol. 190, 258–267. CrossRef CAS PubMed Google Scholar
Dixit, R. (2012). New Phytol. 193, 543–545. CrossRef CAS PubMed Google Scholar
Djinovic Carugo, K., Bañuelos, S. & Saraste, M. (1997). Nature Struct. Biol. 4, 175–179. CAS PubMed Google Scholar
French, S. & Wilson, K. (1978). Acta Cryst. A34, 517–525. CrossRef CAS IUCr Journals Web of Science Google Scholar
Frey, N., Klotz, J. & Nick, P. (2009). Plant Cell Physiol. 50, 1493–1506. CrossRef PubMed CAS Google Scholar
Gimona, M., Djinovic Carugo, K., Kranewitter, W. J. & Winder, S. J. (2002). FEBS Lett. 513, 98–106. Web of Science CrossRef PubMed CAS Google Scholar
Hildebrand, A., Remmert, M., Biegert, A. & Söding, J. (2009). Proteins, 77, Suppl. 9, 128–132. Google Scholar
Howard, J. (1996). Annu. Rev. Physiol. 58, 703–729. CrossRef CAS PubMed Google Scholar
Kabsch, W. (2010). Acta Cryst. D66, 125–132. Web of Science CrossRef CAS IUCr Journals Google Scholar
Klein, M. G., Shi, W., Ramagopal, U., Tseng, Y., Wirtz, D., Kovar, D. R., Staiger, C. J. & Almo, S. C. (2004). Structure, 12, 999–1013. CrossRef PubMed CAS Google Scholar
Klotz, J. & Nick, P. (2012). New Phytol. 193, 576–589. CrossRef CAS PubMed Google Scholar
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. Web of Science CrossRef CAS IUCr Journals Google Scholar
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Oksanen, E., Jaakola, V.-P., Tolonen, T., Valkonen, K., Åkerström, B., Kalkkinen, N., Virtanen, V. & Goldman, A. (2006). Acta Cryst. D62, 1369–1374. Web of Science CrossRef CAS IUCr Journals Google Scholar
Petrášek, J. & Schwarzerová, K. (2009). Curr. Opin. Plant Biol. 12, 728–734. PubMed Google Scholar
Preuss, M. L., Kovar, D. R., Lee, Y.-R. J., Staiger, C. J., Delmer, D. P. & Liu, B. (2004). Plant Physiol. 136, 3945–3955. CrossRef PubMed CAS Google Scholar
Takahashi, K. & Nadal-Ginard, B. (1991). J. Biol. Chem. 266, 13284–13288. CAS PubMed Google Scholar
Umezu, N., Umeki, N., Mitsui, T., Kondo, K. & Maruta, S. (2011). J. Biochem. 149, 91–101. CrossRef CAS PubMed Google Scholar
Wasteneys, G. O. & Galway, M. E. (2003). Annu. Rev. Plant Biol. 54, 691–722. CrossRef PubMed CAS Google Scholar
Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Web of Science CrossRef CAS IUCr Journals Google Scholar
Xu, T., Qu, Z., Yang, X., Qin, X., Xiong, J., Wang, Y., Ren, D. & Liu, G. (2009). Biochem. J. 421, 171–180. Web of Science CrossRef PubMed CAS Google Scholar
Xu, T., Sun, X., Jiang, S., Ren, D. & Liu, G. (2007). J. Biochem. Mol. Biol. 40, 723–730. CrossRef PubMed CAS Google Scholar
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