Poster abstracts

Poster number 15 submitted by Eric Danhart

Conformational and chemical selection by a trans-acting editing domain

Eric M. Danhart (Ohio State Biochemistry Program, The Ohio State University), Marina Bakhtina, William A. Cantara (Department of Chemistry and Biochemistry, The Ohio State University), Alexandra B. Kuzmishin, Brianne L. Sanford (Department of Chemistry and Biochemistry, The Ohio State University), Marija Kosutic, Ronald Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, CMBI, Leopold Franzens University), Yuki Goto, Hiroaki Suga (Department of Chemistry, Graduate School of Science, The University of Tokyo), Karin Musier-Forsyth, Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

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 trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that has been shown to edit Ala-tRNAPro, which is the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla 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 Caulobacter crescentus ProXp-ala, as well as binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a non-hydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation activity. Backbone 15N NMR relaxation experiments revealed ps-ns timescale dynamics of a helix flanking the substrate binding site in free ProXp-ala and when bound to the uncharged microhelix, but dynamics are attenuated upon binding the stably mischarged substrate analog. Computational docking and molecular dynamics simulations are consistent with these findings, and predict a role for the substrate primary α-amine group in substrate recognition. Overall, the results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

Keywords: aminoacyl-tRNA synthetases, trans-editing, conformational selection