crystallization communications\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

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Cloning, purification, crystallization and preliminary X-ray studies of HMO2 from Saccharomyces cerevisiae

aKey Laboratory of Molecular Biology of Infectious Diseases, Chongqing Medical University, 1 Yixueyuan Road, Chongqing 400016, People's Republic of China, and bDepartment of Laboratory Medicine, Chongqing Medical University, 1 Yixueyuan Road, Chongqing 400016, People's Republic of China
*Correspondence e-mail: wangd@ustc.edu

(Received 23 April 2013; accepted 19 November 2013; online 24 December 2013)

The high-mobility group protein (HMO2) of Saccharomyces cerevisiae is a component of the chromatin-remodelling complex INO80, which is involved in double-strand break (DSB) repair. HMO2 can also bind DNA to protect it from exonucleolytic cleavage. Nevertheless, little structural information is available regarding these functions of HMO2. Since determination of three-dimensional structure is a powerful means to facilitate functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of HMO2 from S. cerevisiae are reported. The crystal belonged to space group P222, with unit-cell parameters a = 39.35, b = 75.69, c = 108.03 Å, and diffracted to a resolution of 3.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 3.19 Å3 Da−1.

1. Introduction

High-mobility group box (HMGB) proteins are a set of nuclear non-histone proteins that are found in a variety of eukaryotic species (Bustin, 2001[Bustin, M. (2001). Trends Biochem. Sci. 26, 152-153.]). The mobility proteins are composed of two homologous conserved HMGB domains, Box A and Box B, which bind nonspecifically to the minor groove of DNA. Both of the Box domains contain about 80 amino acids and are contiguous with each other, being located at the N- and C-terminus, respectively, and form an L-­shaped structure with three α-helices (Thomas, 2001[Thomas, J. O. (2001). Biochem. Soc. Trans. 29, 395-401.]; Bianchi & Beltrame, 2000[Bianchi, M. E. & Beltrame, M. (2000). EMBO Rep. 1, 109-114.]; Churchill et al., 2010[Churchill, M. E., Klass, J. & Zoetewey, D. L. (2010). J. Mol. Biol. 403, 88-102.]). Initially regarded as structural components of chromatin, studies over the past decade have drastically changed the view of their cellular role. HMGB proteins have been implicated in transcriptional regulation, DNA repair, recombination, differentiation and extracellular signalling (Thomas, 2001[Thomas, J. O. (2001). Biochem. Soc. Trans. 29, 395-401.]; Scaffidi et al., 2002[Scaffidi, P., Misteli, T. & Bianchi, M. E. (2002). Nature (London), 418, 191-195.]).

Saccharomyces cerevisiae contains several HMGB proteins, including NHP6A/B, HMO1 and HMO2. The single-domain HMGB proteins NHP6A/B participate in transcription by RNA polymerases II and III (Kruppa et al., 2001[Kruppa, M., Moir, R. D., Kolodrubetz, D. & Willis, I. M. (2001). Mol. Cell, 7, 309-318.]; Moreira & Holmberg, 2000[Moreira, J. M. & Holmberg, S. (2000). EMBO J. 19, 6804-6813.]; Lopez et al., 2001[Lopez, S., Livingstone-Zatchej, M., Jourdain, S., Thoma, F., Sentenac, A. & Marsolier, M.-C. (2001). Mol. Cell. Biol. 21, 3096-3104.]; Kassavetis & Steiner, 2006[Kassavetis, G. A. & Steiner, D. F. (2006). J. Biol. Chem. 281, 7445-7451.]). NHP6A folds into an L-shaped pattern which comprises three helices that are held together by two hydrophobic cores for binding to DNA (Murphy et al., 1999[Murphy, F. V., Sweet, R. M. & Churchill, M. E. (1999). EMBO J. 18, 6610-6618.]). HMO1 and HMO2 are similar to mammalian HMGB proteins in containing two HMGB domains (Box A and Box B). HMO1 has been shown to play a role in plasmid maintenance, normal growth, ribosomal RNA transcription and ribosomal protein expression (Lu et al., 1996[Lu, J., Kobayashi, R. & Brill, S. J. (1996). J. Biol. Chem. 271, 33678-33685.]; Gadal et al., 2002[Gadal, O., Labarre, S., Boschiero, C. & Thuriaux, P. (2002). EMBO J. 21, 5498-5507.]; Hall et al., 2006[Hall, D. B., Wade, J. T. & Struhl, K. (2006). Mol. Cell. Biol. 26, 3672-3679.]; Merz et al., 2008[Merz, K., Hondele, M., Goetze, H., Gmelch, K., Stoeckl, U. & Griesenbeck, J. (2008). Genes Dev. 22, 1190-1204.]; Xiao & Grove, 2009[Xiao, L. & Grove, A. (2009). Curr. Genomics, 10, 198-205.]; Berger et al., 2007[Berger, A. B., Decourty, L., Badis, G., Nehrbass, U., Jacquier, A. & Gadal, O. (2007). Mol. Cell. Biol. 27, 8015-8026.]). HMO2, also known as NHP10, is a component of the chromatin-remodelling complex INO80, which is involved in the repair of DNA double-strand breaks (DSBs) and chromatin remodelling (Morrison et al., 2004[Morrison, A. J., Highland, J., Krogan, N. J., Arbel-Eden, A., Greenblatt, J. F., Haber, J. E. & Shen, X. (2004). Cell, 119, 767-775.]). HMO2 has also been reported to be responsible for interaction with the damage-induced phosphorylated histone H2A (γ-H2AX). The inactivation of H2A results in reduced INO80 recruitment to the DSBs, which does not impair chromatin remodelling (Morrison et al., 2004[Morrison, A. J., Highland, J., Krogan, N. J., Arbel-Eden, A., Greenblatt, J. F., Haber, J. E. & Shen, X. (2004). Cell, 119, 767-775.]; Au et al., 2011[Au, T. J., Rodriguez, J., Vincent, J. A. & Tsukiyama, T. (2011). Mol. Cell. Biol. 31, 4454-4463.]; van Attikum et al., 2007[Attikum, H. van, Fritsch, O. & Gasser, S. M. (2007). EMBO J. 26, 4113-4125.]). In addition, HMO2 binds to DNA ends (blunt ends and cohesive ends) and protects DNA ends from exonucleolytic cleavage, which is essential to minimize loss of genetic information before repair (Morrison et al., 2004[Morrison, A. J., Highland, J., Krogan, N. J., Arbel-Eden, A., Greenblatt, J. F., Haber, J. E. & Shen, X. (2004). Cell, 119, 767-775.]; Ray & Grove, 2009[Ray, S. & Grove, A. (2009). Nucleic Acids Res. 37, 6389-6399.]). HMO2 is also thought to arrive early at DSBs in its complex with the INO80 components, suggesting that HMO2 may play a role in DSB repair beyond the recruitment of INO80 (Ray & Grove, 2009[Ray, S. & Grove, A. (2009). Nucleic Acids Res. 37, 6389-6399.]). Recently, Ray and Grove reported that HMO2 preferentially binds to distorted DNA, suggesting that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks (Ray & Grove, 2009[Ray, S. & Grove, A. (2009). Nucleic Acids Res. 37, 6389-6399.]).

Nevertheless, little structural information is available regarding these functions of HMO2. In order to further investigate the biochemical and physiological functions of HMO2, we have initiated the determination of its three-dimensional structure by X-ray crystallography. Here, we report the expression, purification, crystallization and preliminary X-ray crystallographic study of HMO2 from S. cerevisiae. Structure determination will be pursued using experimental phasing methods. Determination of the three-dimensional structure of HMO2 will help in understanding how this widespread protein exerts its multitudinous functions.

2. Materials and methods

2.1. Cloning and expression

The full-length gene encoding HMO2 (NP_010282) was amplified from yeast genomic DNA by the polymerase chain reaction using primers containing NdeI and XhoI restriction sites. Both the PCR products and the vector pET-22b were digested with the NdeI and XhoI restriction enzymes. The ligation mixture was transformed into chemically competent Escherichia coli DH5α cells and the insertion was verified by PCR and DNA sequencing. Recombinant full-length yeast HMO2 with an N-terminal His tag (pET-22b-HMO2) was transformed into chemically competent E. coli B834 cells for expression. Cultures of bacteria carrying pET-22b-HMO2 were grown overnight in 10 ml LB medium supplemented with 20 mg l−1 ampicillin. The bacteria were then used to inoculate 4.0 l LB medium and were cultured at 310 K until the OD600 reached ∼0.5. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. The culture was allowed to grow at 293 K for a further 15 h before the cells were harvested by centrifugation.

2.2. Purification

The cells were resuspended in lysis buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl) and disrupted by sonication. The cell lysate was centrifuged at 15 000g for 40 min at 277 K and the cell debris was discarded. The recombinant protein in the supernatant was applied onto an Ni2+–NTA (Qiagen) column pre-equilibrated with lysis buffer. Nonspecifically bound proteins were washed from the affinity column with 300 ml lysis buffer containing 30 mM imidazole. The recombinant protein was then eluted from the column with 20 ml elution buffer (20 mM Tris–HCl pH 8.0, 200 mM imidazole). The affinity-column eluate was then loaded onto a DEAE Sepharose Fast Flow column (GE Healthcare) pre-equilibrated with 20 mM Tris–HCl pH 8.0 for further purification. The recombinant protein was eluted with a linear gradient of 0–300 mM NaCl in 20 mM Tris–HCl pH 8.0. For crystallization, fractions containing HMO2 were identified using SDS–PAGE and the target fractions were concentrated and buffer-exchanged into the final buffer (5 mM Tris–HCl pH 8.0, 50 mM NaCl) using a Millipore Amicon concentrator with a 5 kDa cutoff membrane. The concentration of the final purified recombinant protein was about 80 mg ml−1 and its purity was determined to be about 95% by SDS–PAGE.

2.3. Crystallization

Preliminary screening of crystallization conditions was performed by the sitting-drop vapour-diffusion method with the Crystal Screen, PEG/Ion and Index kits (Hampton Research). The crystallization experiments consisted of 1.0 µl protein solution (80 mg ml−1) mixed with 1.0 µl reservoir solution and were equilibrated against 100 µl reservoir solution at 293 K. After about 20 d, small crystals of HMO2 were observed using a reservoir condition consisting of 20%(w/v) PEG 3350, 0.2 M ammonium formate pH 6.6. Further optimization of the conditions using PEGs of different molecular weights at various concentrations gave good diffraction-quality crystals with 18–25%(w/v) PEG 3350, 0.2 M ammonium formate in the presence of 0.1 M bis-tris pH 6.3–7.0 by the hanging-drop vapour-diffusion method. Crystals were obtained after 20 d equilibration against the crystallization solution and grew to full size (0.008 × 0.005 × 0.1 mm) in two months (Fig. 1[link]).

[Figure 1]
Figure 1
A crystal of HMO2 of S. cerevisiae grown by the hanging-drop method.

2.4. Data collection and processing

X-ray diffraction data were collected from a single crystal of yeast HMO2 protein using an ADSC Quantum 315r on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF). The crystals were mounted in a nylon-fibre loop and flash-cooled in a liquid-nitrogen gas stream at 110 K without additional cryoprotectant. 180 images were collected at a wavelength of 0.9792 Å using an oscillation angle of 1° and an exposure time of 15 s per frame (Fig. 2[link]). The images were processed and scaled to 3.0 Å using iMosflm (Battye et al., 2011[Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. (2011). Acta Cryst. D67, 271-281.]) and SCALA from the CCP4 suite (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]). The final data-collection and processing statistics are given in Table 1[link].

Table 1
Data-collection and processing statistics

Values in parentheses are for the highest resolution shell.

Space group P222
Unit-cell parameters (Å) a = 39.35, b = 75.69, c = 108.03
Wavelength (Å) 0.9792
Resolution (Å) 40–3.0 (3.16–3.00)
No. of observed reflections 39572
No. of unique reflections 6870
Completeness (%) 99.5 (89.0)
I/σ(I)〉 4.6 (3.6)
Rmerge (%) 10.4 (31.4)
Rmerge = [\textstyle \sum_{hkl}\sum_{i}|I_{i}(hkl)- \langle I(hkl)\rangle|/][\textstyle \sum_{hkl}\sum_{i}I_{i}(hkl)], where Ii(hkl) is the intensity of the ith measurement of an equivalent reflection with indices hkl.
[Figure 2]
Figure 2
An X-ray diffraction pattern of a native crystal of HMO2 of S. cerevisiae recorded using a MAR CCD detector.

3. Results and discussion

A complete diffraction data set was collected to 3.0 Å resolution from a single crystal and the data-collection statistics are reported in Table 1[link]. A total of 39 572 measured reflections in the resolution range 30–3.0 Å were merged into 6870 unique reflections with an Rmerge of 10.4%. Analysis of the diffraction intensities indicated that the most plausible space group was orthorhombic P222, with unit-cell parameters a = 39.35, b = 75.69, c = 108.03 Å. Based on the molecular weight of yeast HMO2 (25.2 kDa) and space group P222, it was assumed that the crystal contained one molecule per asymmetric unit. The assumption gives a VM value of 3.19 Å3 Da−1 and a solvent content of 61.49% (Matthews, 1968[Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497.]). Molecular replacement using AMoRe (Navaza, 2001[Navaza, J. (2001). Acta Cryst. D57, 1367-1372.]), Phaser (McCoy et al., 2005[McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. (2005). Acta Cryst. D61, 458-464.]) and MOLREP (Vagin & Teplyakov, 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) was carried out using the structure of the sequence-nonspecific HMGB protein NHP6A as a search model (PDB entry 1lwm ; Masse et al., 2002[Masse, J. E., Wong, B., Yen, Y.-M., Allain, F. H.-T., Johnson, R. C. & Feigon, J. (2002). J. Mol. Biol. 323, 263-284.]). The model has about 34% sequence identity to the C-terminal domain (amino-acid sequence 90–162) of HMO2 of S. cerevisiae. However, efforts to determine the structure using molecular replacement with NHP6A have proved to be unsuccessful. We are now in the process of producing selenomethionylated HMO2 protein in order to use multi-wavelength anomalous dispersion methods for structure determination.

Acknowledgements

The authors gratefully acknowledge Professor Jianhua He for assistance with native crystal diffraction data collection on beamline BL17U1 of the Shanghai Synchrotron Radiation Facility (SSRF). Financial support for this project was provided by research grants from the Chinese National Natural Science Foundation (grant Nos. 31240082 and 30970563) and the Natural Science Foundation of Chongqing (grant No. 2009BB5413) to DW.

References

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ISSN: 2053-230X
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