Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation
Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation
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DOI：
10.1073/pnas.1907849116
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发表时间：
2019-09-24
影响因子：
11.1
通讯作者：
Zhou, Huan-Xiang
中科院分区：
文献类型：
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作者：
Ghosh, Archishman;Mazarakos, Konstantinos;Zhou, Huan-Xiang
无膜细胞器，对应于蛋白质或蛋白质-RNA混合物液-液分离时的液滴相，调节着无数的细胞功能。细胞使用多种生化信号，如表达水平和翻译后修饰来调节液滴的形成和溶解，但调节机制的物理基础仍然不明确，并且在很大程度上缺乏对其影响的定量评估。我们的计算研究预测，液滴形成蛋白的吸引力强度决定了大分子调节剂是否以及如何促进或抑制LLP。我们使用SH3结构域和富含Pro的基序(SH3(5)和PRM5)的五聚体作为液滴形成蛋白，实验验证了这一预测。测定大范围调节剂浓度范围内相边界的变化和液滴相中的分配系数，既产生了对调节效果的定量测量，也产生了对调节效果的机理理解。观察到了三类典型的调控效应。高浓度的Ficoll 70通过在体相中占据体积，从而将SH3(5)和PRM5置换到液滴相，间接促进SH3(5)-PRM5 LLP。溶菌酶具有中等的分配系数，通过用SH3(5)取代液滴相中较强的SH3(5)-PRM5吸引来抑制LLP。通过与PRM5形成更强的吸引力，低浓度的肝素大量分配到液滴相并促进LLP。这些特征被片状粒子模型的计算结果概括，验证了3类大分子调节剂作为体积排斥促进剂、弱吸引抑制剂和强吸引促进剂的识别。
Membraneless organelles, corresponding to the droplet phase upon liquid-liquid phase separation (LLPS) of protein or protein-RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ill-defined and quantitative assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromolecular regulators promote or suppress LLPS. We experimentally tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH3(5) and PRM5) as droplet-forming proteins. Determination of the changes in phase boundary and the partition coefficients in the droplet phase over a wide range of regulator concentrations yielded both a quantitative measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were observed. Ficoll 70 at high concentrations indirectly promoted SH3(5)-PRM5 LLPS, by taking up volume in the bulk phase and thereby displacing SH3(5) and PRM5 into the droplet phase. Lysozyme had a moderate partition coefficient and suppressed LLPS by substituting weaker attraction with SH3(5) for the stronger SH3(5)-PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concentrations partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromolecular regulators as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.