Cartwright Misti, Parakra Rinky, Oduwole Ayomide, Zhang Fangliang, Deredge Daniel J, Smith Aaron T
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States.
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, United States.
Biochemistry. 2024 Dec 17;63(24):3236-3249. doi: 10.1021/acs.biochem.4c00512. Epub 2024 Dec 6.
Arginyltransferase 1 (ATE1) catalyzes arginylation, an important posttranslational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while posttranslational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse () ATE1. Using size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well those as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR facilitates the formation of a complex between ATE1 and tRNA, adding a new complexity to the ATE1 structure and providing new insights for future studies of ATE1 functions.
精氨酰转移酶1（ATE1）催化精氨酰化反应，这是真核生物中一种重要的翻译后修饰（PTM），在细胞稳态中起着关键作用。ATE1功能的破坏与哺乳动物神经退行性疾病和心血管发育异常有关，而翻译后精氨酰化也与几种重要的人类病毒如严重急性呼吸综合征冠状病毒2（SARS-CoV-2）和人类免疫缺陷病毒（HIV）的活性有关。尽管已知ATE1在哺乳动物细胞功能中具有重要意义，但过去对该酶的生物物理研究主要集中在酵母ATE1上，哺乳动物细胞中精氨酰化的机制仍不清楚。在本研究中，我们试图对小鼠（）ATE1进行结构和生物物理表征。通过尺寸排阻色谱（SEC）、小角X射线散射（SAXS）和氢-氘交换质谱（HDX-MS），并借助AlphaFold建模，我们发现小鼠ATE1在结构上比酵母ATE1更复杂。重要的是，我们的数据表明所有小鼠ATE1剪接变体中都存在一个内在无序区域（IDR）。然而，比较HDX-MS分析表明酵母ATE1没有这样的IDR，这与之前的X射线、冷冻电镜和SAXS分析一致。此外，生物信息学方法揭示，哺乳动物ATE1序列以及绝大多数其他真核生物中的序列，在靠近ATE1 GNAT活性位点折叠处含有一个类似IDR的序列。计算分析表明，该IDR促进了ATE1与tRNA之间复合物的形成，为ATE1结构增加了新的复杂性，并为未来研究ATE1功能提供了新的见解。
