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Structural Basis for Lysidine Formation by ATP Pyrophosphatase Accompanied by a Lysine-Specific Loop and a Trna-Recognition Domain

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Structural Basis for Lysidine Formation by ATP Pyrophosphatase Accompanied by a Lysine-Specific Loop and a Trna-Recognition Domain Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain Kotaro Nakanishi*, Shuya Fukai*, Yoshiho Ikeuchi†, Akiko Soma‡, Yasuhiko Sekine‡, Tsutomu Suzuki†, and Osamu Nureki*§¶ʈ *Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan; †Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; ‡Department of Life Science, College of Science, Rikkyo (St. Paul’s) University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan; §Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan; and ¶RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan Edited by Dieter So¨ll, Yale University, New Haven, CT, and approved April 5, 2005 (received for review February 7, 2005) Lysidine, a lysine-combined modified cytidine, is exclusively lo- acylated by isoleucyl-tRNA synthetase (IleRS), and functions as an cated at the anticodon wobble position (position 34) of eubacterial isoleucine tRNA. Therefore, the single posttranscriptional modifi- Ile Ile tRNA 2 and not only converts the codon specificity from AUG to cation of C34 to lysidine 34 in tRNA 2 concomitantly converts the AUA, but also converts the aminoacylation specificity from recog- codon specificity from AUG to AUA and the amino acid specificity nition by methionyl-tRNA synthetase to that by isoleucyl-tRNA from methionine to isoleucine. synthetase (IleRS). Here, we report the crystal structure of lysidine The gene encoding the enzyme that directly introduces lysine to Ile Ile synthetase (TilS) from Aquifex aeolicus at 2.42-Å resolution. TilS C34 of tRNA 2 was recently identified as tilS (tRNA -lysidine forms a homodimer, and each subunit consists of the N-terminal synthetase) by genetics and proteomics techniques (10). However, dinucleotide-binding fold domain (NTD), with a characteristic cen- the mechanism by which lysidine synthetase (TilS) synthesizes tral hole, and the C-terminal globular domain (CTD) connected by lysidine has not yet been elucidated. Here, we present the crystal a long ␣-helical linker. The NTD shares striking structural similarity structure of TilS from a thermophilic eubacterium, Aquifex aeolicus, BIOCHEMISTRY with the ATP-pyrophosphatase domain of GMP synthetase, which at 2.42 Å. To our knowledge, this study provides the first reported reminds us of the two-step reaction by TilS: adenylation of C34 and structure of TilS; the structure of the E. coli enzyme was only lysine attack on the C2 carbon. Conserved amino acid residues are deposited in the Protein Data Bank as a function-unknown protein clustered around the NTD central hole. Kinetic analyses of the named MesJ. The structure of the catalytic domain of A. aeolicus Ile conserved residues’ mutants indicated that C34 of tRNA 2 is TilS closely resembles that of an ‘‘N-type’’ ATP pyrophosphatase adenylated by an ATP lying across the NTD central hole and that a (PPase), which suggests a possible molecular evolution pathway and lysine, which is activated at a loop appended to the NTD, nucleo- a putative catalytic mechanism of TilS. Extensive mutant analyses philically attacks the C2 carbon from the rear. Escherichia coli TilS based on the atomic structure provide the structural basis for the (called MesJ) has an additional CTD, which may recognize the mechanism of lysidine formation by TilS. Structural and functional Ile tRNA 2 acceptor stem. In contrast, a mutational study revealed comparisons with E. coli TilS revealed that the two TilS enzymes Ile Met that A. aeolicus TilS does not recognize the tRNA acceptor stem but discriminate premodified tRNA 2 from premodified tRNA by recognizes the C29⅐G41 base pair in the anticodon stem. Thus, the strategies similar to that used by IleRS, but in distinct manners. Ile Met two TilS enzymes discriminate tRNA 2 from tRNA by strategies similar to that used by IleRS, but in distinct manners. Materials and Methods Crystallization. The expression vector, pET15-TilS, was constructed crystal structure ͉ isoleucyl-tRNA synthetase ͉ mutagenesis ͉ by cloning the A. aeolicus tilS gene into the plasmid pET-15b RNA modification (Novagen). A culture of E. coli strain BL21 codon plus (DE3), transformed with pET15-TilS, was grown to an OD600 of 0.5, and then the expression of TilS was induced with 1 mM isopropyl he accuracy of protein biosynthesis is ensured by two processes ␤ Tinvolving tRNA: One is the correct attachment of the amino -D-thiogalactopyranoside (IPTG), followed by 20 h of cultivation Ј at 37°C. After centrifugation at 5,000 ϫ g for 30 min, the harvested acid to the 3 terminus of tRNA, which is catalyzed by 20 aminoacyl- ⅐ tRNA synthetases, and the other is codon recognition by the tRNA cells were suspended in 100 mM Tris HCl buffer (pH 7.8) contain- ing 10 mM KCl, 8 mM MgCl2, 10 mM DTT, and 1 mM PMSF and anticodon. The posttranscriptional modification of the anticodon ϫ wobble position (position 34) establishes the codon–anticodon were gently sonicated. After centrifugation at 20,000 g for 30 min, pairing rule to prevent the anticodon from misreading a noncog- the supernatant was heat-treated at 70°C for 30 min, and then the nate codon (1). Several wobble modifications are known to alter the recombinant TilS was purified by chromatography on SP Sepha- codon specificity for precise decoding (2–5). Lysidine is a lysine- rose, Phenyl Superose, and Resource S columns (Amersham Bio- containing cytidine derivative that occurs at the wobble position of sciences). The purified TilS was dialyzed against a crystallization solution composed of 10 mM Tris⅐HCl (pH 8.0) and 10 mM MgCl eubacterial and some organellar AUA codon-specific isoleucine 2 Ile Ile and was concentrated. Crystals of A. aeolicus TilS were grown tRNAs (tRNA 2) (6–8). The premodified tRNA 2 has a CAU anticodon, which also appears in premodified methionine tRNA Met Ile (tRNA ) specific to the AUG codon. The premodified tRNA 2 This paper was submitted directly (Track II) to the PNAS office. reads the AUG codon and is aminoacylated by methionyl-tRNA Abbreviations: IleRS, isoleucyl-tRNA synthetase; TilS, lysidine synthetase; NTD, N-terminal synthetase and thus behaves as a methionine tRNA. A previous in dinucleotide-binding fold domain; CTD, C-terminal globular domain; PPase, pyrophos- vitro microsurgery experiment revealed that the replacement of phatase; rmsd, rms deviation. Ile lysidine 34 by an unmodified C34 in Escherichia coli tRNA 2 Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, drastically reduced the isoleucine-accepting activity but increased www.pdb.org (PDB ID code 1WY5). the methionine-accepting activity (9). Thus, once C34 is modified ʈTo whom correspondence should be addressed. E-mail: [email protected]. Ile to lysidine, the mature tRNA 2 reads the AUA codon, is amino- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501003102 PNAS ͉ May 24, 2005 ͉ vol. 102 ͉ no. 21 ͉ 7487–7492 Downloaded by guest on September 24, 2021 within 2 days at 20°C by the hanging-drop vapor diffusion method. unlabeled L-lysine to the extent that the resulting scintillation counts Drops were prepared by mixing equal volumes ofa5mg͞ml TilS are not below the threshold level. solution and the reservoir solution, containing 0.1 M sodium Ile acetate (pH 4.6) and 3.5 M sodium formate. Selenomethionine- Lysidine Formation Assay of tRNA 2 Mutants. The genes encoding Ile labeled TilS was prepared by the conventional method and was the A. aeolicus tRNA 2 mutants were synthesized by PCR with purified in the same manner as the WT. mutated primers. The subsequent purification steps were the Ile same as those used for the WT A. aeolicus tRNA 2 transcript. Ile Structure Determination and Refinement. The TilS crystals were The lysidine-forming activity of the tRNA 2 mutants were harvested in 0.12 M sodium acetate buffer (pH 4.6) containing 4.2 measured in 50 ␮l of reaction buffer (100 mM Tris⅐HCl, pH ͞ ͞ ͞ M sodium formate and then were sequentially transferred into the 7.8 10 mM MgCl2 10 mM KCl 10 mM DTT) containing 2 mM ␮ Ile ␮ 14 buffers containing 10% and 20% glycerol at 10-min intervals for ATP, 10 M tRNA 2 transcript, 250–4,000 M [U- C] L-lysine, cryoprotection. The crystals were flash-cooled in a nitrogen stream and 0.125 ␮M A. aeolicus TilS. After 2, 5, and 10 min, a 15-␮l aliquot at 100 K. All diffraction data sets were collected at the NW12 was removed. The assay procedures were the same as those used for station at the Photon Factory (Tsukuba, Japan) and were processed the aforementioned kinetic analyses. with the HKL2000 suite (HKL Research, Charlottesville, VA). The data set at the peak wavelength of the multiple wavelength anom- Results and Discussion alous dispersion (MAD) experiment up to 2.63 Å was used to search Overall Structure. We solved the crystal structure of A. aeolicus TilS for the locations of the selenium atoms, by using the program SNB (317 aa) and refined the model to 2.42-Å resolution with an Rcryst (11). Several trials by SNB revealed all eight consistent peaks of 21.8% and an Rfree of 25.3% (Fig. 5, which is published as expected in the asymmetric unit. Subsequent phase refinements supporting information on the PNAS web site). The crystals belong ϭ ϭ were performed with the program SHARP (12), and the model was to the P41212 space group, with unit cell parameters of a b 81.96 ϭ manually built into the electron density maps by using the program Å and c 302.6 Å (Table 2).
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