Structural Insights into the Second Step of RNA-dependent Cysteine Biosynthesis in Archaea: Crystal Structure of Sep-tRNA:Cys-tRNA Synthase from Archaeoglobus fulgidus

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Abstract

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNACys is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNACys and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNACys to Cys-tRNACys. In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 Å resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded β-sheet, which is typical of the pyridoxal 5′-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNACys. On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNACys to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.

Introduction

The universal genetic code of 20 amino acids is interpreted by the canonical set of aminoacyl-tRNA synthetases (aaRSs), which attach specific amino acids to their cognate tRNAs.1 However, methanogenic archaea, such as Methanococcus jannaschii, lack the canonical cysteinyl-tRNA synthetase (CysRS) for the “direct” Cys-tRNACys formation.2 In such an organism, Cys-tRNACys is produced by the “indirect” pathway, in which the non-canonical O-phosphoseryl-tRNA synthetase (SepRS) ligates a non-canonical amino acid, O-phosphoserine (Sep), to tRNACys, and the Sep-tRNA:Cys-tRNA synthase (SepCysS) converts the produced Sep-tRNACys to Cys-tRNACys (Figure 1(a)).3 The SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in such organisms.3 Recently, we determined the structure of the SepRSradical dottRNACysradical dotO-phosphoserine ternary complex from Archaeoglobus fulgidus.4 SepRS forms an α4 tetramer, which binds two tRNACys molecules. The aminoacylation catalytic domain recognizes O-phosphoserine in a unique manner, and the C-terminal anticodon-binding domain recognizes the anticodon loop of tRNACys in a sequence-specific manner. The indirect pathway for Cys-tRNACys formation by SepRS/SepCysS is ancient and may predate the direct pathway by CysRS.4., 5. Therefore, it was hypothesized that cysteine was first recruited to the genetic code by the indirect SepRS/SepCysS pathway.3., 4., 5.

SepCysS is most closely related to the pyridoxal 5′-phosphate (PLP)-dependent cysteine desulfurases, such as CsdB, NifS, and IscS, in terms of its primary sequence. The cysteine desulfurases have the PLP-dependent transferase fold, and convert cysteine to alanine and elemental sulfur by removal of the thiol group (Figure 1(b)–(d))).6., 7. The substrate amino acid (selenocysteine) binds covalently to the PLP-Lys side-chain (Figure 1(d)). The sulfur is donated to the Fe-S cluster of another protein and to modified nucleotides.8 Thus, between SepCysS and the cysteine desulfurases, the catalyzed chemistries are reverse reactions to some extent, and the sizes of the substrates/products vastly differ. The substrate and product molecules are large, with molecular masses of > 24 kDa, in SepCysS (Sep-tRNACys plus an unidentified sulfur donor and Cys-tRNACys, respectively), and small, with molecular masses < 130 Da, in cysteine desulfurases (free cysteine and alanine plus elemental sulfur, respectively). Therefore, although the crystal structures of cysteine desulfurases in various states revealed the substrate recognition mechanisms and suggested some catalytic mechanisms,6., 7., 9. how SepCysS binds Sep-tRNACys and converts it to Cys-tRNACys is unknown because of the lack of tertiary structure information. Actually, the Cys residue that is completely conserved in the CsdB and NifS enzymes and plays a crucial role in the catalysis is absent in the SepCysS enzymes.

Here we determined the crystal structure of A. fulgidus SepCysS at 2.4 Å resolution by the selenomethionine (SeMet) single-wavelength anomalous dispersion (SAD) method. In the structure, SepCysS forms a homodimer and PLP is covalently bound to the conserved lysine side-chain in the active site. The structure provides insight into the SepCysS recognition of Sep-tRNACys.

Section snippets

Overall structure of A. fulgidus SepCysS

The native and SeMet crystals of A. fulgidus SepCysS belong to the space groups P41212 and P6122, respectively. The SeMet structure was solved by the SeMet SAD method, and was refined to 3.0 Å resolution. The native structure was solved by the molecular replacement method, using the SeMet structure as a model, and was refined to 2.4 Å resolution (Figure 2). The asymmetric unit of the native crystal contains two molecules (subunits A and B) of SepCysS, which form a dimer (Figure 2(b)–(d))).

Expression and purification of SepCysS

The gene fragment encoding A. fulgidusSepCysS1 (AF0028) was cloned into the pET26b plasmid (Novagen). The plasmid was transformed into E. coli strain BL21(DE3) codon PLUS (Stratagene). For protein overexpression, the cells were grown to an A600 of 0.6–0.8, and the expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside for an additional 4 h at 37 °C. The cells were harvested and sonicated in 50 mM Tris–HCl buffer (pH 8.0) containing 500 mM NaCl, 5 mM MgCl2, 10 mM dithiothreitol

Acknowledgements

We thank Drs S. Sekine, T. Ito (University of Tokyo), T. Yanagisawa, T. Sengoku, R. Ishii (RIKEN), M. Kawamoto, N. Shimizu, and H. Sakai (JASRI) for their help in data collection at SPring-8. We also thank Dr R. Akasaka (RIKEN) for his help in the analytical ultracentrifugation analysis. 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

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    1

    Present address: S. Yokoyama, Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

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