5VXB

Crystal structure of Caulobacter crescentus ProXp-ala at 1.69 Angstrom

5VXB の概要

• エントリーDOI: 10.2210/pdb5vxb/pdb
• 関連するPDBエントリー: 1VJF
• 分子名称: ProXp-ala (2 entities in total)
• 機能のキーワード: trna trans-editing domain, rna binding protein
• 由来する生物種: Caulobacter crescentus (strain ATCC 19089 / CB15)
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 55711.20

構造登録者

Foster, M.P.,Musier-Forsyth, K.,Nakanishi, K.,Ma, X. (登録日: 2017-05-23, 公開日: 2017-08-09, 最終更新日: 2023-10-04)

主引用文献

Danhart, E.M.,Bakhtina, M.,Cantara, W.A.,Kuzmishin, A.B.,Ma, X.,Sanford, B.L.,Kosutic, M.,Goto, Y.,Suga, H.,Nakanishi, K.,Micura, R.,Foster, M.P.,Musier-Forsyth, K.
Conformational and chemical selection by a trans-acting editing domain.
Proc. Natl. Acad. Sci. U.S.A., 114:E6774-E6783, 2017

PubMed Abstract: Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain -editing proteins. ProXp-ala is a ubiquitous -editing enzyme that edits Ala-tRNA, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNA and mischarged Ala-tRNA is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNA acceptor stem mimic, microhelix, or a nonhydrolyzable mischarged Ala-microhelix substrate analog identified residues important for binding and deacylation. Backbone N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a -editing domain to ensure acceptance of only mischarged Ala-tRNA, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

PubMed: 28768811
DOI: 10.1073/pnas.1703925114

実験手法

X-RAY DIFFRACTION (1.69 Å)