Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Viikinkaari 1, P.O. Box 65, FI-00014 Helsinki, Finland.
Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box 35, FI-40014 Jyvaskyla, Finland.
Biochim Biophys Acta Gen Subj. 2019 Apr;1863(4):749-759. doi: 10.1016/j.bbagen.2019.01.014. Epub 2019 Jan 25.
Ubiquitin-like domains (UbLs), in addition to being post-translationally conjugated to the target through the E1-E2-E3 enzymatic cascade, can be translated as a part of the protein they ought to regulate. As integral UbLs coexist with the rest of the protein, their structural properties can differ from canonical ubiquitin, depending on the protein context and how they interact with it. In this work, we investigate T.th-ubl5, a UbL present in a polyubiquitin locus of Tetrahymena thermophila, which is integral to an ADP-ribosyl transferase protein. Only one other co-occurrence of these two domains within the same protein has been reported.
NMR, multiple sequence alignment, MD simulations and SPR have been used to characterize the structure of T.th-ubl5, identify putative binders and experimentally test the interaction, respectively.
Molecular dynamics simulations showed that T.th-ubl5 is unable to bind the proteasome like ubiquitin due to the lack of the conserved hydrophobic patch. Of other integral UbLs identified by structural and sequence alignment, T.th-ubl5 showed high structural and sequence resemblance with the Ras-binding epitope of FERM UbLs. SPR experiments confirmed that a strong and specific interaction occurs between T.th-ubl5 and T.th-Ras.
Data indicate that T.th-ubl5 does not interact with the proteasome like ubiquitin but acts as a decoy for the recruitment of Ras protein by the ADP-ribosyl transferase domain.
Mono-ADP-ribosylation of Ras proteins is known as a prerogative of bacterial toxins. T.th-ubl5 mediated recruitment of Ras highlights the possibility of an unprecedented post-translational modification with interesting implication for signalling pathways.
除了通过 E1-E2-E3 酶级联反应将泛素样结构域（UbL）翻译后偶联到靶标之外，UbL 还可以作为其应该调节的蛋白质的一部分进行翻译。作为完整的 UbL 与蛋白质的其余部分共存，它们的结构特性可能与典型的泛素不同，这取决于蛋白质的上下文以及它们与之相互作用的方式。在这项工作中，我们研究了 Tetrahymena thermophila 多泛素基因座中的 UbL T.th-ubl5，它是一种 ADP-核糖基转移酶蛋白的组成部分。只有另一种情况是这两个结构域在同一蛋白质中共存。
使用 NMR、多重序列比对、MD 模拟和 SPR 分别来表征 T.th-ubl5 的结构、鉴定潜在的配体并进行实验测试相互作用。
分子动力学模拟表明，由于缺乏保守的疏水性补丁，T.th-ubl5 无法像泛素一样与蛋白酶体结合。在通过结构和序列比对鉴定的其他完整 UbL 中，T.th-ubl5 与 FERM UbL 的 Ras 结合表位具有高度的结构和序列相似性。SPR 实验证实了 T.th-ubl5 和 T.th-Ras 之间发生了强烈和特异性的相互作用。
数据表明，T.th-ubl5 不像泛素那样与蛋白酶体相互作用，但作为 ADP-核糖基转移酶结构域招募 Ras 蛋白的诱饵。
Ras 蛋白的单 ADP-核糖基化被认为是细菌毒素的特权。T.th-ubl5 介导的 Ras 募集突出了一种前所未有的翻译后修饰的可能性，这对信号通路具有有趣的意义。
