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J. Biol. Chem., Vol. 280, Issue 33, 29937-29945, August 19, 2005

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Structural Basis for Non-cognate Amino Acid Discrimination by the Valyl-tRNA Synthetase Editing Domain^*

Ryuya Fukunaga and Shigeyuki Yokoyama¶||**

From the Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, ^¶RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, and ^||RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan

The editing domain of valyl-tRNA synthetase (ValRS) is known to deacylate, or edit, misformed Thr-tRNA^Val (post-transfer editing). Here, we determined the 1.7-Å resolution crystal structure of the Thermus thermophilus ValRS editing domain. A comparison of the structure with the previously reported tRNA complex structure revealed conformational changes of the editing domain upon accommodation of the terminal A76; the "GTG loop" moves to expand the pocket, and the side chain of Phe-264 on the GTG loop rotates to interact with the A76 adenine ring. If these conformational changes did not occur, then C75 and A76 of the tRNA would clash with Phe-264. To elucidate the mechanism of the threonine side-chain recognition, we determined the crystal structure of the editing domain bound with [N-(L-threonyl)-sulfamoyl]adenosine at 1.7-Å resolution. The -OH of the threonyl moiety is recognized by the Lys-270, Thr-272, and Asp-279 side chains, which may reject the cognate valyl moiety. Accordingly, ValRS mutants with an Ala substitution for Lys-270 or Asp-279 synthesized significant amounts of Thr-tRNA^Val. The misproduced Thr-tRNA^Val was hydrolyzed efficiently by the wild-type ValRS, but this post-transfer editing activity was drastically impaired by the Ala substitutions for Lys-270 and Asp-279 and was also decreased by those for Arg-216, Phe-264, and Thr-272. These results indicate that the threonyl moiety and A76 of Thr-tRNA^Val are recognized by the Lys-270, Thr-272, and Asp-279 side chains and by the Phe-264 side chain, respectively, of the ValRS editing domain.

Received for publication, March 10, 2005 , and in revised form, June 8, 2005.

The atomic coordinates and structure factors (codes 1WKA and 1WK9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part 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, and the National Project on Protein Structural and Functional Analyses, MEXT. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists.

^** To whom correspondence should be addressed: Dept. of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4392; Fax: 81-3-5841-8057; E-mail: yokoyama{at}biochem.s.u-tokyo.ac.jp.

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