Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Proc Natl Acad Sci U S A. 2021 Jun 8;118(23). doi: 10.1073/pnas.2104897118.
The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by -GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure-function relationships in other biological condensates.
生物分子凝聚物在调节生物功能中的作用，以及涉及固有无序蛋白区域（IDR）的动态相互作用在其组装中的重要性，越来越受到重视。虽然计算和理论方法为 IDR 相行为提供了重要的见解，但由于缺乏适用于研究这些高度动态大规模相关状态的标准结构生物学工具，通过实验以原子分辨率确定控制凝聚的关键相互作用更加困难。NMR 可以是一种有价值的方法，但凝聚 IDR 的动态和粘性性质带来了挑战。我们使用 CAPRIN1 的 C 端 IDR（607 至 709），这是一种在应激颗粒、P 体和信使 RNA 运输颗粒中发现的 RNA 结合蛋白，开发并应用了各种 NMR 方法来研究凝聚 IDR 状态，以深入了解驱动和调节相分离的相互作用。我们确定了与 CAPRIN1 相互作用的 ATP，这些相互作用可以增强或减少相分离。我们还定量了凝聚的 CAPRIN1 内的特定侧链和骨架相互作用，这些相互作用定义了相分离的关键序列，并且在细胞周期和应激过程中发生的已知的 -GlcNAc 化作用下会减少。为了表征 IDR 凝聚物而开发的这个扩展 NMR 工具包，已经生成了与理解 CAPRIN1 生物学相关的详细相互作用信息，并为相分离的一般模型提供了信息，具有在其他生物凝聚物中阐明动态结构-功能关系的重要潜在未来应用。
