Crystal structures of apo wild‐type M. jannaschii tyrosyl‐tRNA synthetase (TyrRS) and an engineered TyrRS specific for O‐methyl‐L‐tyrosine
Open Access
- 1 May 2005
- journal article
- Published by Wiley in Protein Science
- Vol. 14 (5), 1340-1349
- https://doi.org/10.1110/ps.041239305
Abstract
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 π‐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
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