Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.

Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influences the Nterm conformation. To explore the latter, we used sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm-ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with single-stranded RNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm-ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.

Chirally deuterated benzyl chlorides from benzyl alcohols via hexachloroacetone/polymer-supported triphenylphosphine: synthesis of protected (2S, 3S)-[3-(2)H, (15)N]-tyrosine.

Chirally deuterated benzyl chlorides were prepared using novel, general hexachloroacetone/polymer-supported triphenylphosphine treatment of chirally deuterated benzyl alcohols. Doubly labeled protected tyrosine was obtained in 62% yield with 86% de at the α-carbon and 82% de at the ß-carbon. Key in the synthesis was the alkylation of (15)N-labeled (-)-8-phenylmenthylhippurate with R-(-)-4-triisopropylsilyloxybenzyl-α-d chloride.