New insights into the function of Fascin in actin bundling: A combined theoretical and experimental study

Abstract

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.

Introduction

According to the report of global cancer statistics 2018 (Bray et al., 2018), cancer has been among the top killer diseases in our society, owing to its rapidly growing incidence and high mortality. For cancer patients, tumor metastasis is the primary reason for cause of death (Hall, 2009). Thus, it is important to understand cancer cell migration and the mechanism of tumor metastasis to improve cancer treatment.

On the mechanism exploration of tumor metastasis, the formation of filopodia, as cell surface protrusions, is found to be very important to navigate its surroundings and determine the direction of migration. The filopodia is supported by actin filament network, which are rigid tightly bundling with actin-binding proteins, resulting in the filament bundles (Mattila and Lappalainen, 2008). Notably, Fascin, one of the actin cross-linking proteins, is detected in this process, indicating that Fascin interacts with F-actin (Otto et al., 1979). Also, expression of Fascin is up-regulated in various types of cancers to its overexpression correlates with increased tumor metastasis (Du et al., 2015; Hashimoto et al., 2005; Xie et al., 2010). In our previous studies, it also was found that expression of FSCN1 (gene of Fascin) is up-regulated in esophageal cancer (Wu et al., 2013, 2011). As a result, Fascin is used as a marker in cancer diagnosis and prognosis (Cao et al., 2014; Chen et al., 2017; Machesky and Li, 2010; Tan et al., 2014).

Fascin has a molecular mass of ∼55 kDa and functions as a monomer with fourβ-trefoil domains (Fig. 1a) (Yamashiro-Matsumura and Matsumura, 1985). All the charged groups located on the surface of Fascin are shown as balls in Fig. 1b. Fascin is required to facilitate the cross-linking of actin filaments into straight, compact, rigid bundles (Tilney et al., 1998). Biochemical characterization demonstrates that Fascin functions to generate the well-ordered parallel filaments (DeRosier and Edds, 1980) (Mattila and Lappalainen, 2008).

To understand how Fascin functions, many studies have been carried out to probe the actin-binding sites on Fascin (Chen et al., 2010; Sedeh et al., 2010). Early study by Ono and coworkers proposed that Fascin has two actin-binding sites to enable its bundling activity: one is located at the C terminus (domain 4, Fig. 1a, green) and the other one is at the N terminus (domain 1, Fig. 1a, blue), in which the latter binding site was confirmed by mutation of Ser39 (Ono et al., 1997). Later, Chen and coworkers found that both the N and C termini of Fascin contribute to the same binding site of actin based on their crystal structure (Chen et al., 2010). Importantly, they further confirmed this actin-binding site by experiments of binding macroketone (an inhibitor of actin-bundling) to Fascin and mutagenesis studies of residues His392, Lys471 and Ala488 of Fascin (Chen et al., 2010). In the study by Jansen et al., two multi-site mutants K22E/K43E/R100E/R109E and R271E/K353E/K358E of Fascin decreased the actin bundling activity, indicating that there exist two major actin-binding sites located in domains 1 and 3, respectively (Jansen et al., 2011). Furthermore, Yang and coworkers determined two major actin-binding sites and one possible site based on 100 mutants of Fascin (Yang et al., 2013). One of the two confirmed binding sites are locate between domains 1 and 4, and the other one is between domains 1 and 2 that is opposite to site 1. Additionally, another possible binding site is domain 3.

Further, the relation between structure and function of Fascin has been investigated by mutagenesis studies. The actin-bundling activity of the phosphomimetic S39D fascin mutant is significantly decreased within the phosphomimetic mutant of S39D (Chen et al., 2010; Ono et al., 1997). In a study by Yang and cowokers’ study, comparing the S39D mutant to the crystal structure of wild-type, several apparent conformational changes were found in the S39D mutant, namely inactive conformation (Yang et al., 2013). In addition, our recent study showed that other phosphomimetic mutations of Y23D, S38D and S274D of Fascin could also inhibit the filopodia formation, indicating that phosphorylation might play an important role in actin bundling by Fascin (Zeng et al., 2017).

On the other hand, the cryo-EM structure of actin filaments (F-actin) was also solved with some actin-bundling proteins (ABPs), e.g. tropomyosin, myosin, tropomodulin, cofilin (Tanaka et al., 2018) and filamin (Gurel et al., 2017; Iwamoto et al., 2018; Rao et al., 2014; von der Ecken et al., 2015). It has been found that, for F-actin, subdomains 2 and 4 (SD2 and SD4) of actin 3 (Ac3) interact with the SD3 of Ac5, and the SD3 of Ac3 interact with SD4 of Ac1 in the axial direction (Fig. 2). In the horizontal direction, SD1 and SD2 of Ac3 interact with the SD2 of Ac2 and SD2 of Ac4, respectively. In this way, each G-actin contacts with another 4 actins, and the contact area on each G-actin is ∼3500 Ų, which is ∼1/5 of total surface area of one G-actin (∼16,700 Ų). In addition, the formation of salt bridge on the interfaces between the F-actin and ABPs was found to be important for binding.

Although experimental studies have found some residues on Fascin are essential for the actin bundling, how these residues influence the structure is still not fully understood. Based on our previous studies, phosphorylation plays an important role in decreasing the actin-bundling function of Fascin (Zeng et al., 2017). In this paper, we further examine all the mutations that mimic phosphorylation with MD simulations, i.e. S → D and T → D. We focus mainly on the structural changes following single-site mutation. Finally, five mutants were selected to perform biochemical experiments for validation.