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1 Department of Molecular Biology, 2 Department of Chemistry, and 3 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
(RECEIVED November 25, 2004; FINAL REVISION February 17, 2005; ACCEPTED February 18, 2005)
The Methanococcus jannaschii tRNATyr/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 Å, respectively, for comparison with the published structure of TyrRS complexed with tRNATyr and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257–263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through 
-stacking and hydrogen bonding interactions. Loop 133–143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNATyr. Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133–143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.
Keywords: tyrosyl-tRNA synthetase; crystallography; unnatural amino acid; protein engineering
Abbreviations: TyrRS, tyrosyl-tRNA synthetase • OMeTyr, O-methyl-L-tyrosine • ALS, Advanced Light Source • MR, molecular replacement • CCP4, Collaborative Computational Project 4 • rms deviation, root-mean-square deviation
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041239305.
Reprint requests to: Ian A. Wilson, Department of Molecular Biology, or Peter G. Schultz, Department of Chemistry, The Scripps Research Institute, 10550 Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: wilson{at}scripps.edu or schultz{at}scripps.edu; fax: (858) 784-2980 or (858) 784-9440.
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