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

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Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii

aDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan, bRIKEN Genomic Sciences Center, Japan, and cCellular Signaling Laboratory, RIKEN Harima Institute, Japan
*Correspondence e-mail: yokoyama@biochem.s.u-tokyo.ac.jp

(Received 26 August 2004; accepted 6 September 2004; online 25 September 2004)

All five tRNALeu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS–tRNALeu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNALeu, with a corresponding crystal volume per protein weight of 2.9 Å3 Da−1 and a solvent content of 57.3%.

1. Introduction

Aminoacyl-tRNA synthetases (aaRSs) specifically attach each cognate amino acid to the 3′-end of its cognate tRNA. The high specificity of aaRSs for both the amino acid and tRNA is crucial for accurate protein synthesis. A precise understanding of the high selectivity of aaRSs requires their three-dimensional structures. Leucyl-tRNA synthetase (LeuRS) is a large monomeric class Ia synthetase (its molecular weight is more than 100 kDa). tRNALeu has a characteristic long variable loop. The LeuRS–tRNALeu complex has one of the largest molecular weights of all of the aaRS–tRNA complexes. A common feature of the LeuRSs from bacteria, archaea and eukarya is that they use the discriminator base A73 as one of the identity elements through a base-specific interaction, but do not use anticodon nucleotides as recognition determinants, except for the case of the lower eukarya (Asahara et al., 1993[Asahara, H., Himeno, H., Tamura, K., Hasegawa, T., Watanabe, K. & Shimizu, M. (1993). J. Mol. Biol. 231, 219-229.]; Breitschopf et al., 1995[Breitschopf, K., Achsel, T., Busch, K. & Gross, H. J. (1995). Nucleic Acids Res. 23, 3633-3637.]; Soma et al., 1996[Soma, A., Kumagai, R., Nishikawa, K. & Himeno, H. (1996). J. Mol. Biol. 263, 707-714.], 1999[Soma, A., Uchiyama, K., Sakamoto, T., Maeda, M. & Himeno, H. (1999). J. Mol. Biol. 293, 1029-1038.]). In addition to these common mechanisms, the bacterial, archaeal and eukaryal LeuRSs also use different tRNALeu-recognition mechanisms: the archaeal and higher eukaryal LeuRSs use the long variable loop of tRNALeu as a recognition determinant (Small et al., 1992[Small, I., Marechal-Drouard, L., Masson, J., Pelletier, G., Cosset, A., Weil, J. H. & Dietrich, A. (1992). EMBO J. 11, 1291-1296.]; Breitschopf et al., 1995[Breitschopf, K., Achsel, T., Busch, K. & Gross, H. J. (1995). Nucleic Acids Res. 23, 3633-3637.]; Soma et al., 1999[Soma, A., Uchiyama, K., Sakamoto, T., Maeda, M. & Himeno, H. (1999). J. Mol. Biol. 293, 1029-1038.]), while the bacterial and lower eukaryal LeuRSs do not (Asahara et al., 1993[Asahara, H., Himeno, H., Tamura, K., Hasegawa, T., Watanabe, K. & Shimizu, M. (1993). J. Mol. Biol. 231, 219-229.]; Soma et al., 1996[Soma, A., Kumagai, R., Nishikawa, K. & Himeno, H. (1996). J. Mol. Biol. 263, 707-714.]). Since most of the LeuRSs do not recognize the anticodon and their identity elements are different among species, they are considered to be good engineering targets for genetic code expansion (Anderson & Schultz, 2003[Anderson, J. C. & Schultz, P. G. (2003). Biochemistry, 42, 9598-9608.]).

In addition to LeuRS, the closely related isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) also belong to the class Ia family of aaRSs. These three enzymes have high sequence and structural homology and may have evolved from a common ancestral enzyme (Brown & Doolittle, 1995[Brown, J. R. & Doolittle, W. F. (1995). Proc. Natl Acad. Sci. USA, 92, 2441-2445.]). These three aaRSs have an editing domain (the CP1 domain), which is inserted into the catalytic Rossmann-fold domain and hydrolyzes misactivated (the pre-transfer editing) and mischarged (the post-transfer editing) non-cognate amino acids (Baldwin & Berg, 1966[Baldwin, A. N. & Berg, P. (1966). J. Biol. Chem. 241, 839-845.]; Jakubowski & Goldman, 1992[Jakubowski, H. & Goldman, E. (1992). Microbiol. Rev. 56, 412-429.]; Schmidt & Schimmel, 1995[Schmidt, E. & Schimmel, P. (1995). Biochemistry, 34, 11204-11210.]; Lin et al., 1996[Lin, L., Hale, S. P. & Schimmel, P. (1996). Nature (London), 384, 33-34.]; Hale et al., 1997[Hale, S. P., Auld, D. S., Schmidt, E. & Schimmel, P. (1997). Science, 276, 1250-1252.]; Nureki et al., 1998[Nureki, O., Vassylyev, D. G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T. L., Schimmel, P. & Yokoyama, S. (1998). Science, 280, 578-582.]; Silvian et al., 1999[Silvian, L. F., Wang, J. & Steitz, T. A. (1999). Science, 285, 1074-1077.]; Chen et al., 2000[Chen, J. F., Guo, N. N., Li, T., Wang, E. D. & Wang, Y. L. (2000). Biochemistry, 39, 6726-6731.]; Fukai et al., 2000[Fukai, S., Nureki, O., Sekine, S., Shimada, A., Tao, J., Vassylyev, D. G. & Yokoyama, S. (2000). Cell, 103, 793-803.]; Lincecum et al., 2003[Lincecum, T. L., Tukalo, M., Yaremchuk, A., Mursinna, R. S., Williams, A. M., Sproat, B. S., Van Den Eynde, W., Link, A., Van Calenbergh, S., Grotli, M., Martinis, S. A. & Cusack, S. (2003). Mol. Cell, 11, 951-963.]; Fukunaga et al., 2004[Fukunaga, R., Fukai, S., Ishitani, R., Nureki, O. & Yokoyama, S. (2004). J. Biol. Chem. 279, 8396-8402.]). For pre-transfer editing, the tRNA is necessary, although the reason is still unknown (Baldwin & Berg, 1966[Baldwin, A. N. & Berg, P. (1966). J. Biol. Chem. 241, 839-845.]; Hale et al., 1997[Hale, S. P., Auld, D. S., Schmidt, E. & Schimmel, P. (1997). Science, 276, 1250-1252.]). The crystal structure of LeuRS without tRNA from the bacterium Thermus thermophilus has been determined (Cusack et al., 2000[Cusack, S., Yaremchuk, A. & Tukalo, M. (2000). EMBO J. 19, 2351-2361.]; Lincecum et al., 2003[Lincecum, T. L., Tukalo, M., Yaremchuk, A., Mursinna, R. S., Williams, A. M., Sproat, B. S., Van Den Eynde, W., Link, A., Van Calenbergh, S., Grotli, M., Martinis, S. A. & Cusack, S. (2003). Mol. Cell, 11, 951-963.]). The structure revealed the substrate amino-acid recognition mechanism in the aminoacylation and editing active sites of this eubacterial LeuRS. However, its tRNA recognition and aminoacylation mechanisms, and the role of tRNA in the editing reactions still need to be elucidated. In the present research, we have carried out a crystallization and preliminary X-ray crystallographic analysis of LeuRS from the archaeon Pyrococcus horikoshii, complexed with in vitro-transcribed P. horikoshii tRNALeu.

2. Methods and results

2.1. Purification of in vitro-transcribed P. horikoshii tRNALeu

All five P. horikoshii tRNALeu isoacceptors were transcribed in vitro with T7 RNA polymerase. The transcribed tRNALeu was purified by phenol/chloroform treatment followed by urea–PAGE. The tRNALeu was further purified by anion-exchange chromatography with a ResourceQ column using 20 mM Tris–HCl buffer pH 8.0 containing 8 mM MgCl2 as a starting buffer with a linear gradient of 0–1.0 M NaCl. The tRNALeu-containing fractions were pooled, ethanol-precipitated and dried. The tRNALeu was dissolved in 20 mM Tris–HCl buffer pH 8.0 containing 10 mM MgCl2.

2.2. Overexpression and purification of the native P. horikoshii LeuRS

The full-length P. horikoshii LeuRS is composed of 976 amino-acid residues with a molecular weight of 113 kDa. The recombinant LeuRS was overexpressed in Escherichia coli and purified using the methods used for the C-terminally truncated form of P. horikoshii LeuRS, using a phenyl-Toyopearl column (Tosoh), a ResourceQ column (Amersham Biosiences) and a HiTrap Heparin column (Amersham Biosiences) (Fukunaga & Yokoyama, 2004[Fukunaga, R. & Yokoyama, S. (2004). Acta Cryst. D60, 1916-1918.]). Purified LeuRS was dialyzed against 10 mM Tris–HCl buffer pH 8.0 containing 5 mM MgCl2, 200 µM zinc acetate and 5 mM β-mercaptoethanol and was concentrated to a final concentration of 12 mg ml−1 with a Centricon YM-30 filter (Millipore).

2.3. Crystallization and X-ray data collection

Before crystallization, the tRNALeu was heated at 353 K for 5 min and was gradually cooled to room temperature for refolding. 100 mM AMPPNP (a non-hydrolyzable analogue of ATP) solution was added to the LeuRS to give a final concentration of 1 mM. LeuRS and tRNALeu were mixed in a molar ratio of 1:1.1 with a final LeuRS concentration of 10 mg ml−1, heated at 348 K for 10 min and gradually cooled to room temperature. Combinations of LeuRS and each of the tRNALeu isoacceptors were assessed for cocrystallization. Crystals were obtained in only one crystallization condition from the more than 400 tried for each of the tRNA isoacceptors. Crystals were generated only when the tRNALeu isoacceptor with the anticodon CAA was used. The initial crystallization conditions were refined by optimizing the sample and precipitant concentrations, as well as the molecular ratio between LeuRS and tRNALeu, the pH, temperature, additive reagents and sample/reservoir solution volumes. Thus, crystals suitable for X-ray analysis were obtained in 1 d with the hanging-drop vapour-diffusion method by mixing 1 µl sample solution (in a molar ratio of 1:1.2 for LeuRS:tRNALeu with a final LeuRS concentration of 10 mg ml−1) and 1 µl reservoir solution consisting of 30% 2-propanol, 200 mM trisodium citrate and 100 mM sodium cacodylate buffer pH 6.5 at 293 K (Fig. 1[link]). The mixed sample was equilibrated against 500 µl reservoir solution. To determine whether these were actually LeuRS–tRNALeu complex crystals, they were harvested, washed well, dissolved and examined by SDS–PAGE and urea–PAGE. The results showed that the crystals contained both LeuRS and tRNALeu (data not shown), suggesting that they are LeuRS–tRNALeu complex crystals.

[Figure 1]
Figure 1
A native crystal of the P. horikoshii LeuRS–tRNALeu complex.

For the diffraction data collection, the crystals were directly soaked in a cryoprotectant solution consisting of 30% 2-propanol, 1 mM AMPPNP, 33 mM trisodium citrate, 100 mM sodium cacodylate buffer pH 6.5 and 20% MPD. Native X-ray diffraction data sets were collected using cryocooled (100 K) crystals at BL26B1, SPring-8 (Harima, Japan). The diffraction data were collected using 0.3° oscillations with a crystal-to-detector distance of 320 mm. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K (Fig. 2[link]). The data were indexed and scaled with HKL2000 (Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307-326.]). The data-collection statistics are summarized in Table 1[link]. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is estimated to contain two complexes of LeuRS–tRNALeu, with a corresponding crystal volume per protein weight of 2.9 Å3 Da−1 and a solvent content of 57.3%.

Table 1
Data-collection statistics

Values in parentheses are for the highest resolution shell.

Wavelength (Å) 1.020
Resolution range (Å) 50–3.3 (3.42–3.3)
Measured reflections 219343
Unique reflections 46168
Completeness (%) 92.2 (81.9)
Mean I/σ(I) 18.0 (3.0)
Rmerge (%) 5.1 (18.8)
Rmerge = [\textstyle \sum_{hkl} \sum_{i}|I_{hkli} - \langle I_{hkl}\rangle |/][\textstyle \sum_{hkl} \sum_{i} \langle I \rangle].
[Figure 2]
Figure 2
X-ray diffraction image from a native P. horikoshii LeuRS–tRNALeu crystal. The edge of the detector corresponds to a resolution of 3.2 Å.

Attempts to solve the structure of the P. horikoshii LeuRS–tRNALeu complex by either molecular-replacement procedures or the MAD method using selenomethionine-labelled LeuRS are in progress.

Footnotes

Present address: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.

§Present address: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan.

Acknowledgements

We acknowledge the contribution of Dr M. Masaki in help with data collection at SPring-8. We also thank Drs S. Sekine and T. Sengoku (RIKEN) for fruitful discussion. This work was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the RIKEN Structural Genomics/Proteomics Initiative (RSGI) and the National Project on Protein Structural and Functional Analyses, MEXT. RF was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

References

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First citationAsahara, H., Himeno, H., Tamura, K., Hasegawa, T., Watanabe, K. & Shimizu, M. (1993). J. Mol. Biol. 231, 219–229.  CrossRef CAS PubMed Web of Science Google Scholar
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First citationBrown, J. R. & Doolittle, W. F. (1995). Proc. Natl Acad. Sci. USA, 92, 2441–2445.  CrossRef CAS PubMed Web of Science Google Scholar
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First citationCusack, S., Yaremchuk, A. & Tukalo, M. (2000). EMBO J. 19, 2351–2361.  Web of Science CrossRef PubMed CAS Google Scholar
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First citationFukunaga, R. & Yokoyama, S. (2004). Acta Cryst. D60, 1916–1918.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationHale, S. P., Auld, D. S., Schmidt, E. & Schimmel, P. (1997). Science, 276, 1250–1252.  CrossRef CAS PubMed Web of Science Google Scholar
First citationJakubowski, H. & Goldman, E. (1992). Microbiol. Rev. 56, 412–429.  PubMed CAS Web of Science Google Scholar
First citationLin, L., Hale, S. P. & Schimmel, P. (1996). Nature (London), 384, 33–34.  CrossRef CAS PubMed Web of Science Google Scholar
First citationLincecum, T. L., Tukalo, M., Yaremchuk, A., Mursinna, R. S., Williams, A. M., Sproat, B. S., Van Den Eynde, W., Link, A., Van Calenbergh, S., Grotli, M., Martinis, S. A. & Cusack, S. (2003). Mol. Cell, 11, 951–963.  Web of Science CrossRef PubMed CAS Google Scholar
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First citationOtwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326.  CrossRef CAS Web of Science Google Scholar
First citationSchmidt, E. & Schimmel, P. (1995). Biochemistry, 34, 11204–11210.  CrossRef CAS PubMed Web of Science Google Scholar
First citationSilvian, L. F., Wang, J. & Steitz, T. A. (1999). Science, 285, 1074–1077.  Web of Science CrossRef PubMed CAS Google Scholar
First citationSmall, I., Marechal-Drouard, L., Masson, J., Pelletier, G., Cosset, A., Weil, J. H. & Dietrich, A. (1992). EMBO J. 11, 1291–1296.  PubMed CAS Web of Science Google Scholar
First citationSoma, A., Kumagai, R., Nishikawa, K. & Himeno, H. (1996). J. Mol. Biol. 263, 707–714.  CrossRef CAS PubMed Web of Science Google Scholar
First citationSoma, A., Uchiyama, K., Sakamoto, T., Maeda, M. & Himeno, H. (1999). J. Mol. Biol. 293, 1029–1038.  Web of Science CrossRef PubMed CAS Google Scholar

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