Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion.
The genetic code in a eukaryotic system has been expanded by the engineering of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo-L-tyrosine rather than L-tyrosine. In the present study, we determined the 3-iodo-L-tyrosine- and L-tyrosine-bound structures of the 37V195C mutant of the E. coli TyrRS catalytic domain at 2.0-A resolution. The gamma-methyl group of Val-37 and the sulfur atom of Cys-195 make van der Waals contacts with the iodine atom of 3-iodo-L-tyrosine. The Val-37 and Cys-195 side chains are rigidly fixed by the neighboring residues forming the hydrophobic core of the TyrRS. The major roles of the two mutations are different for the 3-iodo-L-tyrosine-selective recognition in the first step of the aminoacylation reaction (the amino acid activation step): the Y37V mutation eliminates the fatal steric repulsion with the iodine atom, and the Q195C mutation reduces the L-tyrosine misrecognition. The structure of the 37V195C mutant TyrRS complexed with an L-tyrosyladenylate analogue was also solved, indicating that the 3-iodo-L-tyrosine and L-tyrosine side chains are similarly discriminated in the second step (the aminoacyl transfer step). These results demonstrate that the amino acid-binding pocket on the 37V195C mutant is optimized for specific 3-iodo-L-tyrosine recognition.
Kobayashi
Takatsugu
T
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sakamoto
Kensaku
K
Takimura
Tetsuo
T
Sekine
Ryo
R
Kelly
Vincent P
VP
Vincent
Kelly
K
Kamata
Kenji
K
Nishimura
Susumu
S
Yokoyama
Shigeyuki
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D005815:Genetic Code; D005818:Genetic Engineering; D006860:Hydrogen Bonding; D008958:Models, Molecular; D007470:Monoiodotyrosine / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000596:Amino Acids; D011994:Recombinant Proteins; D014448:Tyrosine-tRNA Ligase; D007470:Monoiodotyrosine