6WRQ

Crystal structure of Mj 3-nitro-tyrosine tRNA synthetase (5B) S158C variant bound to 3-nitro-tyrosine

6WRQ の概要

• エントリーDOI: 10.2210/pdb6wrq/pdb
• 関連するPDBエントリー: 4NDA 6WRK 6WRN 6WRT
• 分子名称: Tyrosine--tRNA ligase, GLYCEROL, SODIUM ION, ... (5 entities in total)
• 機能のキーワード: aminoacyl-trna synthetase, 3-nitro-tyrosine, ligase
• 由来する生物種: Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 36763.48

構造登録者

Beyer, J.N.,Hosseinzadeh, P.,Karplus, P.A.,Mehl, R.A.,Cooley, R.B. (登録日: 2020-04-29, 公開日: 2020-07-01, 最終更新日: 2023-10-18)

主引用文献

Beyer, J.N.,Hosseinzadeh, P.,Gottfried-Lee, I.,Van Fossen, E.M.,Zhu, P.,Bednar, R.M.,Karplus, P.A.,Mehl, R.A.,Cooley, R.B.
Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.
J.Mol.Biol., 432:4690-4704, 2020

PubMed Abstract: Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.

PubMed: 32569745
DOI: 10.1016/j.jmb.2020.06.014

実験手法

X-RAY DIFFRACTION (1.85 Å)