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.

中文翻译：

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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。

人赖氨酰-tRNA 合成酶 (hLysRS) 对于 tRNALys 的氨酰化至关重要。高等真核生物 LysRS 具有 N 末端延伸 (Nterm)，先前已证明其可促进高亲和力 tRNA 结合和氨酰化。这种真核生物特异性附加结构域在 hLysRS 核定位中也发挥着关键作用，从而促进 hLysRS 的非规范功能。该结构本质上是无序的，因此仍然很难表征。先前的研究结果与 Nterm 结构域在核酸结合后经历构象转变为有序结构是一致的。在本研究中，我们使用 NMR 来研究 RNA 的类型以及相邻反密码子结合域 (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 连接来产生分段标记的串联结构域蛋白 Nterm-ACB。在没有 RNA 的情况下，无论 ACB 是否附着，Nterm 仍然处于无序状态。无论是单独使用还是连接到 ACB 上，Nterm 结构都不受单链 RNA 滴定的影响。Nterm 结构域的中心区域在用含有双链区域的 RNA 发夹滴定 Nterm 和 Nterm-ACB 时采用 α-螺旋结构。Nterm 与 RNA 发夹的结合导致 CD 光谱变化，与诱导的螺旋结构一致。NMR 和荧光各向异性表明 Nterm 与发夹 RNA 的结合很弱，但在共价连接到 ACB 后，结合亲和力显着增加。我们得出结论，ACB 结构域促进诱导拟合构象变化并赋予高亲和力 RNA 发夹结合，