Gavrilov Yulian, Hagai Tzachi, Levy Yaakov
Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel.
Protein Sci. 2015 Oct;24(10):1580-92. doi: 10.1002/pro.2688. Epub 2015 Jun 9.
Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.
泛素化是蛋白质最常见的翻译后修饰之一，在其他细胞过程中介导蛋白质的调控降解。关于蛋白质泛素化机制的一个基本问题是泛素是否以及如何影响被修饰蛋白质的生物物理性质。对于某些系统，研究表明泛素在连接位点内的位置相当灵活，且泛素与其底物没有特异性相互作用。然而，研究发现多聚泛素化可通过折叠态和解折叠态热力学性质的改变，以位点特异性方式降低被修饰蛋白质的热稳定性。在本研究中，我们使用详细的原子模拟来关注泛素化对被修饰蛋白质天然结构的分子效应。作为模型，我们使用了Ubc7，它是一种E2酶，其体内泛素化过程已得到充分表征且已知会导致降解。我们发现，尽管泛素部分与Ubc7之间缺乏特异性直接相互作用，但泛素化会降低底物Ubc7蛋白某些区域的构象灵活性，从而降低其熵并使其不稳定。对于将赖氨酸48连接的四聚泛素连接到用于体内降解的位点的系统，观察到最强的去稳定作用。这些结果揭示了折叠态构象熵的变化如何调节蛋白质天然态的稳定性。总体而言，我们的结果表明泛素化可改变折叠态下被连接蛋白质的生物物理性质，并且在某些蛋白质中，不同的泛素化位点会导致不同的生物物理结果。我们提出多聚泛素对底物的这种去稳定作用与该修饰所执行的功能有关，特别是通过蛋白酶体降解对蛋白质半衰期的调控。
