5VXB

Crystal structure of Caulobacter crescentus ProXp-ala at 1.69 Angstrom

Summary for 5VXB

• Entry DOI: 10.2210/pdb5vxb/pdb
• Related: 1VJF
• Descriptor: ProXp-ala (2 entities in total)
• Functional Keywords: trna trans-editing domain, rna binding protein
• Biological source: Caulobacter crescentus (strain ATCC 19089 / CB15)
• Total number of polymer chains: 3
• Total formula weight: 55711.20

Authors

Foster, M.P.,Musier-Forsyth, K.,Nakanishi, K.,Ma, X. (deposition date: 2017-05-23, release date: 2017-08-09, Last modification date: 2023-10-04)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.69 Å)