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

Journal logoSTRUCTURAL BIOLOGY
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ISSN: 2053-230X
Volume 65| Part 10| October 2009| Pages 1030-1034

Crystallization and crystal-packing studies of Chlorella virus deoxyuridine triphosphatase

aDepartment of Chemistry, University of Nebraska-Lincoln, NE 68583-0304, USA, bCenter for Biotechnology, University of Nebraska-Lincoln, NE 68588-0666, USA, and cSchool of Biological Sciences, University of Nebraska-Lincoln, NE 68583-0118, USA
*Correspondence e-mail: hmoriyama2@unl.edu

(Received 10 July 2009; accepted 27 August 2009; online 25 September 2009)

The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298 K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65 Å. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 Å. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68 Å and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45 Å, γ = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase.

1. Introduction

The optimal temperature for enzyme activity is determined by a trade-off between structural stability and chemical reactivity and is often similar to the physiological conditions of the source organism (Vieille & Zeikus, 2001[Vieille, C. & Zeikus, G. J. (2001). Microbiol. Mol. Biol. Rev. 65, 1-43.]). Previously, we have used the monomeric protein ribonuclease A (RNase; Kadonosono et al., 2003[Kadonosono, T., Chatani, E., Hayashi, R., Moriyama, H. & Ueki, T. (2003). Biochemistry, 42, 10651-10658.]; Chatani et al., 2002[Chatani, E., Hayashi, R., Moriyama, H. & Ueki, T. (2002). Protein Sci. 11, 72-81.]) and the homodimeric protein 3-isopropylmalate dehydro­genase (IMD; Imada et al., 1991[Imada, K., Sato, M., Tanaka, N., Katsube, Y., Matsuura, Y. & Oshima, T. (1991). J. Mol. Biol. 222, 725-738.]; Moriyama et al., 1995[Moriyama, H., Onodera, K., Sakurai, M., Tanaka, N., Kirino-Kagawa, H., Oshima, T. & Katsube, Y. (1995). J. Biochem. (Tokyo), 117, 408-413.]; Hori et al., 2000[Hori, T., Moriyama, H., Kawaguchi, J., Hayashi-Iwasaki, Y., Oshima, T. & Tanaka, N. (2000). Protein Eng. 13, 527-533.]) to study the relationship between temperature and enzyme activity. These studies showed that the size of the active-site cavity influences enzyme stability. In RNase, the internal size of the active-site cavity is inversely correlated with stability (Chatani et al., 2002[Chatani, E., Hayashi, R., Moriyama, H. & Ueki, T. (2002). Protein Sci. 11, 72-81.]). In IMD, a single mutation, Gly240Ala, within the active-site cavity resulted in loss of enzyme thermostability; the optimal temperature decreased owing to the loss of hydrophobic interactions of the sub­units with the enlarged internal cavity (Moriyama et al., 1995[Moriyama, H., Onodera, K., Sakurai, M., Tanaka, N., Kirino-Kagawa, H., Oshima, T. & Katsube, Y. (1995). J. Biochem. (Tokyo), 117, 408-413.]). More recently, we have studied the relationship between temperature and enzyme activity using the homotrimeric enzyme deoxyuridine pyro­phosphatase (dUTPase; Zhang et al., 2005[Zhang, Y., Moriyama, H., Homma, K. & Van Etten, J. L. (2005). J. Virol. 79, 9945-9953.]; Bajaj & Moriyama, 2007[Bajaj, M. & Moriyama, H. (2007). Acta Cryst. F63, 409-411.]).

dUTPase (EC 3.6.1.23) is a ubiquitous enzyme (Cedergren-Zeppezauer et al., 1992[Cedergren-Zeppezauer, E. S., Larsson, G., Nyman, P. O., Dauter, Z. & Wilson, K. S. (1992). Nature (London), 355, 740-743.]) that catalyzes the hydrolysis of dUTP to diphosphate and dUMP, the substrate for thymidylate synthetase in dTTP biosynthesis (Phan et al., 2001[Phan, J., Koli, S., Minor, W., Dunlap, R. B., Berger, S. H. & Lebioda, L. (2001). Biochemistry, 40, 1897-1902.]). The first dUTPase crystal structure to be resolved was that of Escherichia coli (Cedergren-Zeppezauer et al., 1992[Cedergren-Zeppezauer, E. S., Larsson, G., Nyman, P. O., Dauter, Z. & Wilson, K. S. (1992). Nature (London), 355, 740-743.]). Since then, the structure of human dUTPase (Mol et al., 1996[Mol, C. D., Harris, J. M., McIntosh, E. M. & Tainer, J. A. (1996). Structure, 4, 1077-1092.]) has been identified, as have those of human pathogens, including tuberculosis (Varga et al., 2008[Varga, B., Barabas, O., Takacs, E., Nagy, N., Nagy, P. & Vertessy, B. G. (2008). Biochem. Biophys. Res. Commun. 373, 8-13.]; Vertessy & Toth, 2009[Vertessy, B. G. & Toth, J. (2009). Acc. Chem. Res. 42, 97-106.]). dUTPase controls the pyrimidine-nucleotide pool balance and hence is involved in cell death (Mashiyama et al., 2008[Mashiyama, S. T., Hansen, C. M., Roitman, E., Sarmiento, S., Leklem, J. E., Shultz, T. D. & Ames, B. N. (2008). Anal. Biochem. 372, 21-31.]). Recently, dUTPase has received further clinical attention as compromising dUTPase activity with oxaliplatin (via tumor protein p53) has been found to increase the efficiency of fluorouracil (5-FU) inhibition of thymidylate synthetase (Wilson et al., 2009[Wilson, P. M., Fazzone, W., LaBonte, M. J., Lenz, H. J. & Ladner, R. D. (2009). Nucleic Acids Res. 37, 78-95.]). Physicochemical studies (Takacs et al., 2004[Takacs, E., Grolmusz, V. K. & Vertessy, B. G. (2004). FEBS Lett. 566, 48-54.]) and structural insights into drug design have been reported (Samal et al., 2007[Samal, A., Schormann, N., Cook, W. J., DeLucas, L. J. & Chattopadhyay, D. (2007). Acta Cryst. D63, 571-580.]).

The algal Chlorella viruses PBCV-1 and IL-3A have dUTPases with different optimal temperatures (323 K for the former and 310 K for the latter; Zhang et al., 2005[Zhang, Y., Moriyama, H., Homma, K. & Van Etten, J. L. (2005). J. Virol. 79, 9945-9953.]). We previously generated Mu-22 dUTPase by mutating two IL-3A residues to the corresponding residues from PBCV-1 dUTPase (Zhang et al., 2005[Zhang, Y., Moriyama, H., Homma, K. & Van Etten, J. L. (2005). J. Virol. 79, 9945-9953.]). The presence of these two mutations (Glu81Ser and Thr84Arg), creating the derivative Mu-22 dUTPase, led to an increase in the optimal temperature of the enzyme to 328 K (Zhang et al., 2005[Zhang, Y., Moriyama, H., Homma, K. & Van Etten, J. L. (2005). J. Virol. 79, 9945-9953.]). Here, we conduct structural studies of dUTPases from Chlorella viruses in order to elucidate the molecular mechanisms behind the differences in optimal temperature.

2. Methods and results

The IL-3A, Mu-22 and PBCV-1 dUTPases had a T7-epitope tag (MASMTGGQQMGRGSEF) at the N-terminus plus a histidine tag (LEHHHHHH) at the C-terminus (Zhang et al., 2005[Zhang, Y., Moriyama, H., Homma, K. & Van Etten, J. L. (2005). J. Virol. 79, 9945-9953.]). These enzymes were placed in a solution containing 50 mM NaH2PO4, 50 mM NaCl and 100 mM imidazole. Initial crystallization trials were performed using previously reported conditions (Vertessy & Toth, 2009[Vertessy, B. G. & Toth, J. (2009). Acc. Chem. Res. 42, 97-106.]). Briefly, the hanging-drop vapor-diffusion method was per­formed using a MembFac matrix (Hampton Research, Aliso Viejo, California, USA) with a 2 µl drop and 500 µl reservoir at room temperature (approximately 298 K). An initial ragged rock-like crystal was found in a droplet containing 50 mM sodium citrate tribasic dihydrate pH 5.6 and 10%(w/v) 2-propanol. After refining the crystallization conditions, we obtained polyhedral single crystals by using a 8.2 mg ml−1 protein matrix and a reservoir containing 12%(w/v) polyethylene glycol (PEG) 4000, 10%(w/v) 2-propanol and 0.3 M sodium citrate pH 5.65 (Fig. 1[link]a). All crystallizations were performed for one week.

[Figure 1]
Figure 1
Crystallization of T7-His-tagged dUTPases. (a) Apo IL-3A. (b) Soaking of apo IL-3A in dUDP. (c) Cocrystallization of IL3A with EDTA. (d) Cocrystallization of IL3A with dUDP. (e) Macroseeding crystallization of Mu-22 using apo IL-3A. (f) Cocrystallization of Mu-22 dUTPase with dUDP.

The tagged IL-3A dUTPase was subjected to diffraction studies after being soaked in 2.5 mM deoxyuridine diphosphate (dUDP; Fig. 1[link]b) as previously described for plant dUTPase (Bajaj & Moriyama, 2007[Bajaj, M. & Moriyama, H. (2007). Acta Cryst. F63, 409-411.]). We used the flash-freezing method for crystal mounting with 20%(v/v) glycerol, 12%(w/v) PEG 4000, 10%(v/v) 2-­propanol and 0.23 M sodium citrate pH 8.0. A complete data set was collected from a single crystal using Cu Kα radiation (wavelength 1.542 Å) from a generator operating at 40 kV and 20 mA. The diffraction images were indexed, integrated and scaled using HKL-2000 (HKL Research Inc.; Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307-326.]) and found to diffract to a resolution of up to 3.0 Å. Crystal parameters and data-collection and structure-refinement statistics are summarized in Table 1[link].

Table 1
Summary of crystallographic data

dUTPase T7-His-tagged IL-3A Gly-Ser IL-3A Gly-Ser Mu-22
PDB code 3ca9 3c2t 3c3i
Crystallization Soaking with dUDP Cocrystallization with dUDP Cocrystallization with dUDP
Space group P213 P213 P63
Unit-cell parameters (Å, °) a = 105.65 a = 105.68 a = 132.07, c = 53.45, γ = 120.0
Interactions Head-to-head Head-to-head Side
Resolution 50.0–3.0 50–3.0 50–2.4
Total reflections 84696 80123 88640
Unique reflections 8218 8112 20383
Completeness (%) 99.9 99.4 96.6
Redundancy 10.3 9.9 4.4
Rmerge 0.10 0.10 0.06
I/σ(I) 13.4 13.2 21.4
Refinement (Å) 30.50–3.0 33.42–3.0 33.61–3.0
Completeness (%) 100.0 97.8 99.0
Rwork 0.232 0.245 0.193
Rfree 0.307 0.285 0.263
Baverage2) 30.0 30.3 28.0
No. of subunits in ASU 1 + 1 1 + 1 1 + 3
Residues determined      
 Chain A −2–125 1–125 2–128
 Chain B 0–125 1–124 2–137
 Chain C     2–137
 Chain D     2–131
Protein atoms 1909 1864 3939
†Observed intermonomer interactions in crystals. Details are given in Fig. 3[link].

Preliminary structural analysis was performed using the molecular-replacement method with chain A of human dUTPase as a template (PDB code 1q5h ; Mol et al., 1996[Mol, C. D., Harris, J. M., McIntosh, E. M. & Tainer, J. A. (1996). Structure, 4, 1077-1092.]) and assuming the presence of two monomers per asymmetric unit. The structure was determined after repetitive calculations and visual inspections and contained 127 and 125 visible residues out of 143 for monomers A and B, respectively. The N-terminal and C-terminal residues were not visible in the electron-density map. The refined structure yielded an R factor of 0.23 for data from 30.50 to 3.0 Å resolutiom. This structure has been deposited in the Protein Data Bank with PDB code 3ca9 . In addition, to remove the divalent cations, crystallization of apo IL-­3A dUTPase was performed in ethylenediaminetetraacetic acid (EDTA) and the same crystals were obtained (Fig. 1[link]c).

Cocrystallization of IL-3A dUTPase with dUDP was performed using a droplet containing 9.0 mg ml−1 protein and 2.6 mM dUDP and a reservoir containing 11%(w/v) PEG 4000, 10%(w/v) 2-pro­panol and 0.3 M sodium citrate pH 5.65. This yielded only thin stacked prismatic crystals (Fig. 1[link]d). Diffraction experiments on the X6A beamline of the National Synchrotron Light source showed that the crystal belonged to the orthorhombic space group P222, with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 Å, and diffracted to 3.5 Å resolution.

Although crystallization of apo Mu-22 dUTPase was not success­ful, tiny polyhedral crystals were formed after macroseeding with apo cubic IL-3A dUTPase (Fig. 1[link]e). However, these crystals were unstable and repeatedly disappeared during a time frame of hours, making them useful only for studying initial crystal growth. Cocrystallization of Mu-22 dUTPase and dUDP was performed using a droplet containing 0.57 mM (approximately 8.2 mg ml−1) protein and 1.14 mM dUDP and a reservoir containing 10%(w/v) PEG 4000, 10%(w/v) 2-propanol and 0.3 M sodium citrate pH 5.6. This yielded thin stacked rectangular crystals (Fig. 1[link]f) that gave only a few diffraction spots after 30 s of exposure on the X6A beamline.

PBCV-1 dUTPase crystallization was unsuccessful because of aggregation. N-terminally His-tagged IL-3A and Mu-22 dUTPases were constructed and subjected to crystallization. However, heavy precipitation was observed throughout the initial crystallization screens. IL-3A, Mu-22 and PBCV-1 dUTPases were then expressed as glutathione S-transferase (GST) fusion proteins using the pGEX-2T vector (GE Healthcare, Piscataway, New Jersey, USA). This allowed higher levels of protein expression in E. coli. Purification using GST-Sepharose column chromatography followed by thrombin cleavage produced glycyl-seryl-dUTPases (143 amino acids). IL-3A and Mu-22 dUTPase protein solutions were concentrated to 0.7 mM (approximately 10 mg ml−1) protein. dUDP at 2 mM in a buffer containing 50 mM sodium phosphate, 50 mM NaCl and 1.0%(v/v) glycerol pH 8.0 was added. The hanging-drop vapor-diffusion method was used to crystallize the dUTPases. IL-3A dUTPase formed thin rod-shaped crystals after 16 h when a reservoir solution containing 20%(w/v) PEG 1450 and 5 mM MgCl2 was used. Mu-22 dUTPase formed thick short hexagonal crystals after 14 h when a reservoir solution containing 15%(w/v) PEG 1500 was used. Thrombin cleavage of the PBCV-1 fusion protein resulted in multiple fragments and no pure protein.

Preliminary X-ray studies indicated that the needle-like IL-3A dUTPase crystals consisted of two monomers and belonged to the cubic space group P213, with unit-cell parameter a = 105.68 Å (Table 1[link]; Fig. 2[link]a; PDB code 3c2t ). Coupled Mu-22 dUTPase crystals consisted of four monomers and belonged to the hexagonal space group P63, with unit-cell parameters a = 132.07, c = 53.45 Å, γ = 120° (Table 1[link]; Fig. 2[link]b; PDB code 3c3i ).

[Figure 2]
Figure 2
Crystals of glycyl-seryl dUTPases from IL-3A (a) and Mu-22 (b).

Arabidopsis dUTPase crystals consist of one asymmetric trimer (PDB code 2p9o ; Bajaj & Moriyama, 2007[Bajaj, M. & Moriyama, H. (2007). Acta Cryst. F63, 409-411.]), as do those of human dUTPase (PDB code 1q5h ; Mol et al., 1996[Mol, C. D., Harris, J. M., McIntosh, E. M. & Tainer, J. A. (1996). Structure, 4, 1077-1092.]); these crystals belong to the orthorhombic space groups P212121 and P21212, respectively. Two neighboring trimers in a unit cell make a head-to-tail contact along the xy plane in the Arabidopsis and human dUTPase crystals. In human dUTPase, one of the three His-tag tails contributes to this inter-trimer interaction, whereas the other two His tags were not visible. Both the T7-His-tagged IL-3A dUTPase and the glycyl-seryl-tagged protein have the same crystal packing, but this packing differs from those of Arabidopsis and human dUTPases. This consists of two trimers aligned head-to-head in a 21 manner held to two monomers by polar contacts (Fig. 3[link]a). The two trimer arrays are connected via a short antiparallel β-sheet between residues 120 and 125.

[Figure 3]
Figure 3
Monomer contacts in an asymmetric unit. (a) IL-3A dUTPase. Two monomers were seen. Dotted trapezoids represent a trimer molecule. Solid figures labeled A and B represent the two monomers (chains). Polar contacts between monomers A and B (dotted circle) are listed on the right. (b) Mu-22 dUTPase. One trimer and one monomer were seen. Solid figures labeled ABC and D represent the monomers. Monomer A and the trimer composed of monomers BC and D are connected by dUDP (small solid circle). Polar contacts between monomer A and trimer BCD (dotted circle) are listed on the right. Dotted squares indicate the locations of the C-termini.

Although we used very similar crystallization conditions, Mu-22 dUTPase had different monomer-to-monomer interactions from those of IL-3A dUTPase owing to the mutation Thr84Arg (Fig. 3[link]). As a result, glycyl-seryl-tagged Mu-22 dUTPase consists of a dUDP molecule located between a monomer and trimer. The dUDP provides intermonomer interactions via polar contacts (Fig. 3[link]b). Instead of the head-to-head interaction found in IL-3A, side interactions were formed in Mu-22 (Fig. 3[link]b). The dUTPase molecules form a blunted pyramid shape and thus the molecules do not fill the space perfectly, creating a central hole in the crystal.

Footnotes

Current address: Pathology and Microbiology Department, University of Nebraska Medical Center, Omaha, NE 68198-6495, USA.

Acknowledgements

The dUTPases used in this study were generously donated by Drs James Van Etten and Yuanzheng Zhang at the University of Nebraska-Lincoln (UNL). We thank Dr Mamoru Yamanishi at UNL for his computational assistance. We would like to thank Dr Vivian Stojanoff and her team members at the National Synchrotron Light Source, Brookhaven National Laboratory for their technical contributions. This work was supported in part by Nebraska Tobacco Settlement Biomedical Research Development Funds (HM). A portion of the X-ray diffraction studies were carried out at the X6A beamline of the National Synchrotron Light Source, Brookhaven National Laboratory.

References

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Journal logoSTRUCTURAL BIOLOGY
COMMUNICATIONS
ISSN: 2053-230X
Volume 65| Part 10| October 2009| Pages 1030-1034
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