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

Sheng Liu,Maryanne Refaei,Shuohui Liu,Aaron Decker,Jennifer M Hinerman,Andrew B Herr,Mike Howell,Karin Musier-Forsyth,Pearl Tsang

doi:10.1074/jbc.ra120.013852

Abstract

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.

Highlights

The NMR-estimated Kd for N-terminal extension (Nterm) binding to anticodon stem-loop (ACSL) is 45.8 mM, whereas the Kd determined for Nterm–anticodon-binding domain (ACB) binding by fluorescence anisotropy (FA) is 54 nM, corresponding to an 851fold improvement in binding affinity
ACB alone bound to these RNAs with intermediate affinity, with a 3-fold tighter binding to the ACSL relative to the acceptor stem minihelix (ACC), as expected (Table 1)
Consistent with previous studies, we found that human lysyl-tRNA synthetase (hLysRS) Nterm alone is mostly unstructured and monomeric in solution but adopts helical structure upon interaction with a hairpin RNA (ACSL) derived from tRNALys [37]

Summary

Introduction

One general trend that is apparent from these data is that attachment of the ACB domain significantly improves binding of Nterm to these RNAs. The NMR-estimated Kd for Nterm binding to ACSL is 45.8 mM, whereas the Kd determined for Nterm–ACB binding by FA is 54 nM, corresponding to an 851fold improvement in binding affinity. Consistent with previous studies, we found that hLysRS Nterm alone is mostly unstructured and monomeric in solution but adopts helical structure upon interaction with a hairpin RNA (ACSL) derived from tRNALys [37].

Results

Conclusion