Fascin, one of actin bundling proteins, plays an important role in the cross-linking of actin filaments (F-actin). Phosphorylation of Fascin is an important posttranslational modification to affect its structure and function. For example, a phosphomimetic mutation of Fascin-S39D decrease its bundling ability with F-actin significantly. In this paper, we studied the actin-bundling activity of Fascin by using molecular dynamics (MD) simulations and biochemical methods. All single-site mutations from serine/threonine to aspartic acid were mimicked by MD simulations. For five mutants (S146D, S156D, S218D, T239D and S259D), the mutated residues in domain 2 of Fascin were found to form salt-bridge interactions with an adjacent residue, indicating that mutations of these residues could potentially reduce actin-bundling activity. Further, F-actin-bundling assays and immunofluorescence technique showed S146D and T239D to have a strong effect on Fascin bundling with F-actin. Finally, we show that single-site mutations do not change the general shape of Fascin, but local structures near the mutated residues in Fascin-S146D and T239D become unstable, thereby affecting the ability of Fascin to bind with F-actin. These findings suggest that targeting domain 2 of Fascin would be very useful for the drug design. In addition, our study indicates that MD simulation is a useful method to screening which residues on Fascin are important.

肌成束蛋白是肌动蛋白成束蛋白之一，在肌动蛋白丝（F-肌动蛋白）的交联中发挥着重要作用。 Fascin 的磷酸化是影响其结构和功能的重要翻译后修饰。例如，Fascin-S39D 的磷酸模拟突变显着降低了其与 F-肌动蛋白的结合能力。在本文中，我们利用分子动力学（MD）模拟和生化方法研究了Fascin的肌动蛋白成束活性。 MD 模拟模拟了从丝氨酸/苏氨酸到天冬氨酸的所有单位点突变。对于五个突变体（S146D、S156D、S218D、T239D 和 S259D），发现 Fascin 结构域 2 中的突变残基与相邻残基形成盐桥相互作用，表明这些残基的突变可能会降低肌动蛋白捆绑活性。此外，F-肌动蛋白捆绑测定和免疫荧光技术表明，S146D和T239D对肌成束蛋白与F-肌动蛋白的捆绑具有很强的影响。最后，我们发现单位点突变不会改变Fascin的总体形状，但Fascin-S146D和T239D中突变残基附近的局部结构变得不稳定，从而影响Fascin与F-肌动蛋白结合的能力。这些发现表明，靶向肌成束蛋白的结构域 2 对于药物设计非常有用。此外，我们的研究表明MD模拟是筛选Fascin上哪些残基重要的有用方法。