Vimer Shay, Ben-Nissan Gili, Morgenstern David, Kumar-Deshmukh Fanindra, Polkinghorn Caley, Quintyn Royston S, Vasil'ev Yury V, Beckman Joseph S, Elad Nadav, Wysocki Vicki H, Sharon Michal
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.
Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, Israel.
ACS Cent Sci. 2020 Apr 22;6(4):573-588. doi: 10.1021/acscentsci.0c00080. Epub 2020 Apr 10.
Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast () and mammals (rat - , rabbit - and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.
直系同源蛋白复合物在不同生物体中负责等效功能。然而，在进化过程中，每个生物体都会进行适应性调整，以满足其生理需求以及其生态位所带来的环境挑战。这种选择压力导致蛋白复合物的结构多样性，而这种多样性往往难以确定，尤其是在缺乏高分辨率结构的情况下。在此，我们描述了一种基于天然质谱（MS）工具的多级实验方法，用于阐明高度相关的蛋白复合物之间的结构保存情况和差异。20S蛋白酶体是一种重要的蛋白质降解机制，作为我们的模型系统，我们研究了从不同生物体中分离出的五种复合物。我们发现，在整个进化过程中，从古细菌原核复合物到酵母（）和哺乳动物（大鼠 - 、兔子 - 和人类 - HEK293细胞）中的真核20S蛋白酶体，蛋白酶体在大小和稳定性方面都有所增加。大鼠和兔子20S蛋白酶体的天然MS结构特征，此前缺乏高分辨率的三维结构，与人类复合物的结构特征高度相似。使用冷冻电子显微镜单颗粒分析，我们能够获得大鼠20S蛋白酶体的高分辨率结构，从而验证基于MS的结果。我们的研究还表明，酵母复合物而非哺乳动物中的复合物，在大小上最大，并且表现出最大程度的动力学稳定性。此外，我们还鉴定出PSMA7亚基的一种新的蛋白异构体，它存在于大鼠和兔子的复合物中，据我们所知，此前尚未有过相关描述。总之，我们的策略能够阐明彼此高度相似的蛋白复合物的独特结构特性，这一框架不仅对直系同源蛋白复合物有效，对其他高度相关的蛋白组装体也同样有效。
