Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Molecular Medicine, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):28795-28805. doi: 10.1073/pnas.2008122117. Epub 2020 Nov 2.
Endeavoring toward a transferable, predictive coarse-grained explicit-chain model for biomolecular condensates underlain by liquid-liquid phase separation (LLPS) of proteins, we conducted multiple-chain simulations of the N-terminal intrinsically disordered region (IDR) of DEAD-box helicase Ddx4, as a test case, to assess roles of electrostatic, hydrophobic, cation-π, and aromatic interactions in amino acid sequence-dependent LLPS. We evaluated three different residue-residue interaction schemes with a shared electrostatic potential. Neither a common hydrophobicity scheme nor one augmented with arginine/lysine-aromatic cation-π interactions consistently accounted for available experimental LLPS data on the wild-type, a charge-scrambled, a phenylalanine-to-alanine (FtoA), and an arginine-to-lysine (RtoK) mutant of Ddx4 IDR. In contrast, interactions based on contact statistics among folded globular protein structures reproduce the overall experimental trend, including that the RtoK mutant has a much diminished LLPS propensity. Consistency between simulation and experiment was also found for RtoK mutants of P-granule protein LAF-1, underscoring that, to a degree, important LLPS-driving π-related interactions are embodied in classical statistical potentials. Further elucidation is necessary, however, especially of phenylalanine's role in condensate assembly because experiments on FtoA and tyrosine-to-phenylalanine mutants suggest that LLPS-driving phenylalanine interactions are significantly weaker than posited by common statistical potentials. Protein-protein electrostatic interactions are modulated by relative permittivity, which in general depends on aqueous protein concentration. Analytical theory suggests that this dependence entails enhanced interprotein interactions in the condensed phase but more favorable protein-solvent interactions in the dilute phase. The opposing trends lead to only a modest overall impact on LLPS.
为了努力建立一个可转移、可预测的粗粒显式链模型，用于研究蛋白质液-液相分离（LLPS）下的生物分子凝聚物，我们对 DEAD -box 解旋酶 Ddx4 的 N 端无规卷曲区域（IDR）进行了多链模拟，作为一个测试案例，以评估静电、疏水、阳离子-π 和芳香相互作用在氨基酸序列依赖性 LLPS 中的作用。我们评估了三种不同的残基-残基相互作用方案，它们具有共享的静电势。常见的疏水性方案或增强了精氨酸/赖氨酸-芳香阳离子-π 相互作用的方案都不能一致地解释野生型、电荷混淆型、苯丙氨酸到丙氨酸（FtoA）和精氨酸到赖氨酸（RtoK）突变型 Ddx4 IDR 的现有实验 LLPS 数据。相比之下，基于折叠球状蛋白结构之间的接触统计的相互作用再现了总体实验趋势，包括 RtoK 突变体的 LLPS 倾向大大降低。模拟和实验之间的一致性也在 P-颗粒蛋白 LAF-1 的 RtoK 突变体中发现，这强调了，在某种程度上，重要的 LLPS 驱动的 π 相关相互作用体现在经典统计势中。然而，还需要进一步阐明，特别是关于苯丙氨酸在凝聚物组装中的作用，因为对 FtoA 和酪氨酸到苯丙氨酸突变体的实验表明，LLPS 驱动的苯丙氨酸相互作用明显弱于常见统计势所假设的。蛋白质-蛋白质静电相互作用由相对介电常数调制，相对介电常数通常取决于水相中的蛋白质浓度。分析理论表明，这种依赖性导致凝聚相中的蛋白质间相互作用增强，但在稀相中的蛋白质-溶剂相互作用更有利。相反的趋势导致对 LLPS 的影响仅略有增加。
