Fascin,一种肌动蛋白捆绑蛋白,在肌动蛋白丝(F-actin)的交联中起着重要作用。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的磷模拟突变显着降低了其与F-肌动蛋白的捆绑能力。在本文中,我们通过分子动力学(MD)模拟和生化方法研究了Fascin的肌动蛋白捆绑活性。通过MD模拟模拟模拟从丝氨酸/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。Further,F-肌动蛋白捆绑测定和免疫荧光技术显示S146D和T239D对Fascin与F-肌动蛋白捆绑具有强烈影响。最后,我们表明,单位点突变不会改变Fascin的一般形状,但是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Fascin的靶向结构域2对于药物设计将是非常有用的。此外,我们的研究表明,MD模拟是筛选Fascin上重要残基的有用方法。
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.