School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea.
Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
J Am Chem Soc. 2021 Aug 18;143(32):12509-12523. doi: 10.1021/jacs.1c00811. Epub 2021 Aug 6.
Prevalent in diverse protein interactomes, intrinsically disordered proteins or regions (IDPs or IDRs) often drive assembly of higher-order macromolecular complexes, using multiple target-binding motifs. Such IDP hubs are suggested to process various cellular signals and coordinate relevant biological processes. However, the mechanism of assembly and functional regulation of IDP hubs remains elusive due to the challenges in dissecting their intricate protein-protein interaction networks. Here we present basic thermodynamic models for the assembly of simple IDP hubs with multiple target proteins, constructing partition functions from fundamental binding parameters. We combined these basic functions to develop advanced thermodynamic models to analyze the assembly of the Nup153 hubs interacting with multiple karyopherin β1 (Kap) molecules, critical components of nucleocytoplasmic transport. The thermodynamic analysis revealed a complex organization of the Kap binding sites within the C-terminal IDR of Nup153 including a high-affinity 1:1 interaction site and a series of low-affinity sites for binding of multiple Kaps with negative cooperativity. The negative cooperativity arises from the overlapping nature of the low-affinity sites where Kap occupies multiple dipeptide motifs. The quantitative dissection culminated in construction of the Nup153 hub ensemble, elucidating how distribution among various Kap-bound states is modulated by Kap concentration and competing nuclear proteins. In particular, the Kap occupancy of the IDR can be fine-tuned by varying the location of competition within the overlapping sites, suggesting coupling of specific nuclear processes to distinct transport activities. In general, our results demonstrate the feasibility and a potential mechanism for manifold regulation of IDP functions by diverse cellular signals.
在各种蛋白质互作组中普遍存在的无规卷曲蛋白质或区域（IDPs 或 IDRs）通常使用多个靶标结合基序来驱动更高阶的大分子复合物的组装。这些 IDP 中心体被认为可以处理各种细胞信号，并协调相关的生物学过程。然而，由于在剖析其复杂的蛋白质-蛋白质相互作用网络方面存在挑战，IDP 中心体的组装和功能调节机制仍然难以捉摸。在这里，我们提出了具有多个靶标蛋白的简单 IDP 中心体组装的基本热力学模型，从基本的结合参数构建了分配函数。我们将这些基本功能结合起来，开发了先进的热力学模型来分析与多个核孔蛋白 β1（Kap）分子相互作用的 Nup153 中心体的组装，Kap 分子是核质转运的关键组成部分。热力学分析揭示了 Nup153 中 C 端 IDR 内 Kap 结合位点的复杂组织，包括一个高亲和力的 1:1 相互作用位点和一系列具有负协同性的用于结合多个 Kap 的低亲和力位点。负协同性源于低亲和力位点的重叠性质，其中 Kap 占据多个二肽基序。定量剖析最终构建了 Nup153 中心体集合，阐明了 Kap 浓度和竞争核蛋白如何调节各种 Kap 结合状态之间的分布。特别是，通过改变重叠位点内竞争的位置，可以精细调节 IDR 中的 Kap 占有率，这表明特定核过程与不同的转运活性之间存在偶联。总的来说，我们的结果证明了通过各种细胞信号对 IDP 功能进行多方面调节的可行性和潜在机制。
