TY - JOUR
T1 - Structural Basis for Substrate Recognition by the Editing Domain of Isoleucyl-tRNA Synthetase
AU - Fukunaga, Ryuya
AU - Yokoyama, Shigeyuki
N1 - Funding Information:
We thank Drs S. Sekine (University of Tokyo), T. Sengoku, T. Yanagisawa (RIKEN), and M. Konno (University of Ochanomizu); M. Yamamoto and H. Tanida (RIKEN) for their help in data collection at SPring-8. 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. R.F. was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists.
PY - 2006/6/16
Y1 - 2006/6/16
N2 - In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNAIle, in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 Å resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120° and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180°. The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T. thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.
AB - In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNAIle, in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 Å resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120° and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180°. The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T. thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.
KW - amino acid
KW - aminoacyl-tRNA synthetase
KW - editing
KW - isoleucine
KW - valine
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U2 - 10.1016/j.jmb.2006.04.025
DO - 10.1016/j.jmb.2006.04.025
M3 - Article
C2 - 16697013
AN - SCOPUS:33744899308
SN - 0022-2836
VL - 359
SP - 901
EP - 912
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 4
ER -