Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Viikinkaari 1, P.O. Box 65, FI-00014 Helsinki, Finland.
Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
Biochim Biophys Acta Gen Subj. 2021 May;1865(5):129844. doi: 10.1016/j.bbagen.2021.129844. Epub 2021 Jan 11.
Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5).
We solved the X-ray structures of BIL2 in both the inactive and unprecedented, zinc-induced active, forms. Then, we characterized by mass spectrometry the BUBL splicing products in the absence and in the presence of T.thRas-GTPase. Finally, we investigated the effect of ubiquitination on T.thRas-GTPase by molecular dynamics simulations.
The structural analysis demonstrated that zinc-induced conformational change activates protein splicing. Moreover, mass spectrometry characterization of the splicing products shed light on the possible function of BIL2, which operates as a "single-ubiquitin-dispensing-platform", allowing the conjugation, via isopeptide bond formation (K(εNH)-C-ter), of ubl4 to either ubl5 or T.thRas-GTPase. Lastly, we demonstrated that T.thRas-GTPase ubiquitination occurs in proximity of the nucleotide binding pocket and stabilizes the protein active state.
We demonstrated that BIL2 is activated by zinc and that protein splicing induced by this intein does not take place through classical or aminolysis mechanisms but via formation of a covalent isopeptide bond, causing the ubiquitination of endogenous substrates such as T.thRas-GTPase.
In this "enzyme-free" ubiquitination mechanism the isopeptide formation, which canonically requires E1-E2-E3 enzymatic cascade and constitutes the alphabet of ubiquitin biology, is achieved in a single, concerted step without energy consumption.
内含子是一种介入蛋白，已知其能执行蛋白剪接。反应的结果是产生一个内含子结构域和一个无内含子的蛋白质，后者没有插入的痕迹。BIL2 是嗜热四膜虫多泛素基因座（BUBL）的一部分，其中两个缺乏 C+1 亲核体的细菌内含子样（BIL）结构域被两个独立的泛素样结构域（ubl4/ubl5）包围。
我们分别解析了 BIL2 在非活性和前所未有的锌诱导活性两种状态下的 X 射线结构。然后，我们通过质谱法在缺乏和存在 T.thRas-GTPase 的情况下对 BUBL 剪接产物进行了表征。最后，我们通过分子动力学模拟研究了泛素化对 T.thRas-GTPase 的影响。
结构分析表明，锌诱导的构象变化激活了蛋白剪接。此外，对剪接产物的质谱分析揭示了 BIL2 的可能功能，它作为一个“单泛素分配平台”，通过形成异肽键（K(εNH)-C-ter），允许 ubl4 与 ubl5 或 T.thRas-GTPase 结合。最后，我们证明 T.thRas-GTPase 的泛素化发生在核苷酸结合口袋附近，并稳定了蛋白质的活性状态。
我们证明了 BIL2 被锌激活，并且这种内含子诱导的蛋白剪接不是通过经典或氨解机制发生的，而是通过形成共价异肽键，导致内源性底物如 T.thRas-GTPase 的泛素化。
在这种“无酶”泛素化机制中，异肽键的形成，通常需要 E1-E2-E3 酶级联反应，并且构成泛素生物学的字母表，在一个单一的、协调的步骤中完成，不需要能量消耗。
