Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.
Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.
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发夹 RNA 诱导真核特异性赖氨酰-tRNA 合成酶延伸的构象变化以及相邻反密码子结合域的作用。
DOI：
10.1074/jbc.ra120.013852
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Tsang,Pearl
中科院分区：
文献类型：
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作者：
Liu,Sheng;Refaei,Maryanne;Liu,Shuohui;Decker,Aaron;Hinerman,JenniferM;Herr,AndrewB;Howell,Mike;Musier-Forsyth,Karin;Tsang,Pearl
人赖氨酰-tRNA合成酶（hLysRS）对于tRNALys的氨酰化是必需的。高等真核生物LysRS具有N-末端延伸（Nterm），以前显示有利于高亲和力tRNA结合和氨酰化。该真核生物特异性附加结构域在hLysRS核定位中也起关键作用，从而促进hLysRS的非经典功能。该结构本质上是无序的，因此仍然很难表征。先前研究的发现与Nterm结构域在核酸结合后经历构象转变为有序结构一致。在这项研究中，我们使用NMR来研究RNA的类型以及相邻的反密码子结合结构域（ACB）的存在如何影响Nterm构象。为了探索后者，我们使用分选酶A连接来产生区段标记的串联结构域蛋白Nterm-ACB。在RNA的情况下，Nterm保持无序，无论ACB附件。无论是单独还是与ACB连接时，Nterm结构都不受单链RNA滴定的影响。用含有双链区域的RNA发夹滴定Nterm和Nterm-ACB时，Nterm结构域的中心区域采用α-螺旋结构。Nterm与RNA发夹的结合导致CD光谱位移与诱导的螺旋结构一致。NMR和荧光各向异性显示Nterm与发夹RNA的结合较弱，但与ACB共价连接后结合亲和力显著增加。我们得出结论，ACB结构域促进诱导适合构象变化，并赋予高亲和力RNA发夹结合，这可能是有利的LysRS与各种不同的结合伙伴的功能相互作用。
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.