Ramirez Dominique A, Hough Loren E, Shirts Michael R
Department of Biochemistry, University of Colorado Boulder, Boulder CO, 80309.
Department of Physics and BioFrontiers Institute, University of Colorado Boulder, Boulder CO, 80309.
bioRxiv. 2023 Jul 24:2023.05.31.543124. doi: 10.1101/2023.05.31.543124.
Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions which support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and has implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins.
液-液相分离（LLPS）被认为是无膜细胞器形成的主要驱动力。这类细胞器的例子包括中心体、中央纺锤体和应激颗粒。最近，研究表明卷曲螺旋（CC）蛋白，如中心体蛋白中心粒外周蛋白、spd-5和中心体素，可能具有液-液相分离的能力。CC结构域具有使其成为液-液相分离驱动因素的物理特性，但它们是否在这一过程中发挥直接作用尚不清楚。我们开发了一个粗粒度模拟框架来研究CC蛋白的液-液相分离倾向，其中支持液-液相分离的相互作用仅源于CC结构域。我们使用这个框架表明，CC结构域的物理特性足以驱动蛋白质的液-液相分离。该框架专门设计用于研究CC结构域的数量以及CC结构域的多聚化状态如何影响液-液相分离。我们表明，只有两个CC结构域的小模型蛋白也能发生相分离。每个蛋白质的CC结构域数量增加到四个会在一定程度上增加液-液相分离倾向。我们证明，形成三聚体和四聚体的CC结构域比形成二聚体的卷曲螺旋具有显著更高的液-液相分离倾向，这表明多聚化状态对液-液相分离的影响比每个蛋白质的CC结构域数量更大。这些数据支持了CC结构域作为蛋白质液-液相分离驱动因素的假设，并对未来鉴定中心体和中央纺锤体蛋白的液-液相分离驱动区域的研究具有启示意义。
