PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of actafjournal home pagethis articleInternational Union of Crystallographysearchsubscribearticle submission
 
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 July 1; 64(Pt 7): 639–640.
Published online 2008 June 11. doi:  10.1107/S1744309108016928
PMCID: PMC2443980

Crystallization and crystallographic analysis of human NUDT16

Abstract

Human NUDT16, a decapping enzyme belonging to the Nudix superfamily, plays a pivotal role in U8 snoRNA stability. Recombinant NUDT16 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals, which diffracted to 2.10 Å resolution, belonged to space group P212121, with unit-cell parameters a = 44.47, b = 79.32, c = 97.20 Å. The Matthews coefficient and the solvent content were calculated to be 1.92 Å3 Da−1 and 35.84%, respectively, for two molecules per asymmetric unit.

Keywords: NUDT16, Nudix superfamily

1. Introduction

Members of the superfamily of Nudix hydrolases are widespread in bacteria, viruses, eukaryotes and archaea. They catalyze the hydrolysis of a nucleoside diphosphate linked to another moiety X. These enzymes are characterized by a highly conserved 23-amino-acid Nudix motif, GX 5EX 7REUXEEXGU, where U is an aliphatic or hydrophobic residue (Bessman et al., 1996 [triangle]). This motif is involved in catalysis and the binding of Mg2+, Mn2+ or other divalent cations (Mildvan et al., 2005 [triangle]). The substrates of these enzymes are NpnN, (d)NTP and capped RNAs (Ito et al., 2005 [triangle]; Fisher et al., 2004 [triangle]; Hori et al., 2005 [triangle]). NUDT16 is the human homologue of Xenopus laevis X29 protein, which binds and decaps U8 snoRNA and other nuclear RNAs in vitro (Ghosh et al., 2004 [triangle]; Tomasevic & Peculis, 1999 [triangle]). The varying divalent metal can determine the RNA-substrate specificity and the efficiency of the decapping activity of NUDT16 and X29 (Peculis et al., 2007 [triangle]). The results of Peculis and coworkers showed that U8 snoRNA is involved in the processing of 5.8S and 28S rRNAs and that the decapping of U8 snoRNA leads to its degradation. Thus, X29 may be involved in the maturation of the large ribosomal subunit through regulating the stability of U8 snoRNA (Ghosh et al., 2004 [triangle]). NUDT16 shares 51% identity with X29, but its structure and function is unclear.

Several structures of Nudix hydrolases have been determined, e.g. those of X. laevis X29 (Scarsdale et al., 2006 [triangle]), Schizosaccharomyces pombe Dcp2p (She et al., 2006 [triangle]) and Escherichia coli ADPRase (Gabelli et al., 2001 [triangle]). Structural similarity is only observed in the Nudix domain of these proteins, which is the catalytic centre responsible for catalysis and the binding of divalent cations. There are several important regions that determine the substrate specificity of Nudix hydrolases (Mildvan et al., 2005 [triangle]). The nucleotide diphos­phatase activity of X29 is metal-dependent and requires the presence of a significant part of the RNA substrate, but no known RNA-binding motif has been observed. However, from its crystal structure we can observe a plausible RNA-binding channel on the positive face of the protein created by a striking surface dipolarity and unique structural features (Scarsdale et al., 2006 [triangle]). A high-resolution crystal structure would provide much valuable information on the catalytic mechanism of NUDT16 and other Nudix hydrolases.

In this study, we report the cloning and expression of NUDT16 in E. coli. Purified recombinant NUDT16 protein was crystallized using the hanging-drop vapour-diffusion method. Finally, we performed a preliminary crystallographic analysis of human NUDT16.

2. Materials and methods

2.1. Cloning and expression

Human kidney cDNA library was used as a template to amplify the full-length coding sequence of NUDT16 with the specific primers 5′-GGAATTCCATATGATGGCTGGTGCTCGTCGTCTGGAGCTA­GGCGAG-3′ (forward) and 5′-ATAGTTTAGCGGCCGCCTAGTG­GTGGTGGTGGTGATGAGCTGGAATC-3′ (reverse). The amplified NUDT16 DNA fragment was inserted into pET30a+ vector with NdeI/NcoI sites. The positive clone was confirmed by direct DNA sequencing and double-enzyme digestion.

The plasmid was transformed into E. coli strain BL21 (DE3) competent cells. For NUDT16 expression, a single colony was incubated in Luria–Bertani (LB) medium with 50 µg ml−1 kanamycin sulfate (Amresco USA) for overnight growth at 310 K. The overnight culture was then transferred into 2 l fresh LB medium. When the culture density (OD600) reached about 0.7, IPTG (isopropyl β-d-1-thiogalactopyranoside; Sigma, USA) was added to a final concentration of 0.1 mM and the culture was kept at 310 K for 4 h. The expressed NUDT16 contains a C-terminal His tag introduced by the primer.

2.2. Protein purification

The cells were harvested and resuspended in chilled lysis buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 5% glycerol) and homogenized by sonication. The supernatant was mixed with Ni–NTA beads (Qiagen) for 2 h at 277 K. The loaded beads were washed three times with lysis buffer. NUDT16 was then eluted with lysis buffer supplemented with 150 mM imidazole. The elution fractions were further purified using a Superdex 75 gel-filtration column (GE Healthcare). The protein peak was collected and concentrated to about 20 mg ml−1 by ultrafiltration (Millipore) for crystallization.

2.3. Crystallization and X-ray diffraction analysis

Cystallization attempts were carried out with Crystal Screens I and II (Hampton Research) at 291 and 277 K using the hanging-drop vapour-diffusion method. The drops were prepared by mixing 1 µl protein solution and 1 µl reservoir solution and were equilibrated against 200 µl reservoir solution.

X-ray diffraction data were collected in-house on a Rigaku MicroMax007 rotating-anode X-ray generator operated at 40 kV and 20 mA (Cu Kα; λ = 1.5418 Å) equipped with an R-AXIS VII++ image-plate detector. A single crystal was transferred to cryoprotectant solution containing 20% glycerol and 0.6 M imidazole pH 7.0, picked up in a nylon loop and flash-cooled in a cold nitrogen-gas stream at 100 K using an Oxford Cryosystem. DENZO and SCALEPACK from the HKL-2000 program suite were used to index, integrate and scale the data (Otwinowski & Minor, 1997 [triangle]).

3. Results and discussion

Human NUDT16 was expressed in E. coli as a soluble protein with a His tag at the C-terminus. The purified protein migrated with a molecular weight of 22 kDa on SDS–PAGE. Recombinant NUDT16 possesses the activity of decapping U8 snoRNA (data not shown).

NUDT16 crystals grew at 291 K after two weeks from a solution consisting of 1.0 M imidazole pH 7.0. Diffraction data were collected to 2.10 Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 44.47, b = 79.32, c = 97.20 Å. Detailed data statistics are given in Table 1 [triangle]. As in the case of X. laevis X29 (Peculis et al., 2004 [triangle]), the crystallographic asymmetric unit con­tains two molecules. The Matthews coefficient and solvent content were calculated to be 1.92 Å3 Da−1 and 35.84% (Matthews, 1968 [triangle]), respectively.

Table 1
Summary of data-collection statistics

The molecular-replacement method was performed with the program AMoRe (Navaza, 1994 [triangle]) using the crystal structure of X. laevis X29 as a search model (PDB code 1u20). The R working and R free values fell to 28% and 34%, respectively, and refinement is in progress. Attempts are also being made to crystallize the NUDT16–substrate complex.

Acknowledgments

This work was completed in the laboratory of Professor George F. Gao at the Institute of Microbiology, Chinese Academy of Sciences and was supported by the National Natural Science Foundation of China (NSFC 30670091).

References

  • Bessman, M. J., Frick, D. N. & O’Handley, S. F. (1996). J. Biol. Chem.271, 25059–25062. [PubMed]
  • Fisher, D. I., Cartwright, J. L., Harashima, H., Kamiya, H. & McLennan, A. G. (2004). BMC Biochem.5, 7. [PMC free article] [PubMed]
  • Gabelli, S. B., Bianchet, M. A., Bessman, M. J. & Amzel, L. M. (2001). Nature Struct. Biol.8, 467–472. [PubMed]
  • Ghosh, T., Peterson, B., Tomasevic, N. & Peculis, B. A. (2004). Mol. Cell, 13, 817–828. [PubMed]
  • Hori, M., Fujikawa, K., Kasai, H., Harashima, H. & Kamiya, H. (2005). DNA Repair (Amst.), 4, 33–39. [PubMed]
  • Ito, R., Hayakawa, H., Sekiguchi, M. & Ishibashi, T. (2005). Biochemistry, 44, 6670–6674. [PubMed]
  • Matthews, B. W. (1968). J. Mol. Biol.33, 491–497. [PubMed]
  • Mildvan, A. S., Xia, Z., Azurmendi, H. F., Saraswat, V. M., Legler, P., Massiah, M. A., Gabelli, S. B., Bianchet, M. A., Kang, L.-W. & Amzel, L. M. (2005). Arch. Biochem. Biophys.433, 129–143. [PubMed]
  • Navaza, J. (1994). Acta Cryst. A50, 157–163.
  • Otwinowski, Z. & Minor, W. (1997). Methods Enzymol.276, 307–326.
  • Peculis, B. A., Reynolds, K. & Cleland, M. (2007). J. Biol. Chem.282, 24792–24805. [PubMed]
  • Peculis, B. A., Scarsdale, J. N. & Wright, H. T. (2004). Acta Cryst. D60, 1668–1669. [PubMed]
  • Scarsdale, J. N., Peculis, B. A. & Wright, H. T. (2006). Structure, 14, 331–343. [PubMed]
  • She, M., Decker, C. J., Chen, N., Tumati, S., Parker, R. & Song, H. (2006). Nature Struct. Mol. Biol.13, 63–70. [PMC free article] [PubMed]
  • Tomasevic, N. & Peculis, B. (1999). J. Biol. Chem.274, 35914–35920. [PubMed]

Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography