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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. [ABSTRACT FROM AUTHOR]