Mechanism of Actin Filament Bundling by Fascin

Abstract

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ␤-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ␤-trefoil domains 1 and 3. The site in ␤-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ␤-trefoil-3 is related by pseudo-2-fold symmetry to that in ␤-trefoil-1. The two sites are ϳ5 nm apart, resulting in a distance between actin filaments in the bundle of ϳ8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization

“…Others and we previously solved the X-ray crystal structure of fascin 35,45,46 . Based on the crystal structure, we generated 100 mutations of surface-exposed residues on fascin, and determined ARTICLE the actin-bundling and actin-binding activity of these 100 mutants 44 .…”

Molecular Mechanism of Fascin Function in Filopodial Formation

“…S2, B and C). The crystal structures of wild-type fascin have also been solved by two other laboratories under different conditions (PDB codes 1DFC and 3P53) (22,24). Comparisons of these structures show that the two molecules in an asymmetric unit of PDB structure code 1DFC are similar to each other and to Chain A of our PDB structure 3LLP (supplemental Fig.…”

“…To understand why fascin (among all actin bundling or cross-linking proteins) is uniquely selected for filopodial formation, we have investigated the biochemical and biophysical properties of fascin. We and others have recently solved the x-ray crystal structure of fascin (22)(23)(24) (Fig. 1A).…”

“…Elevated levels of fascin have been found in metastatic tumors and are correlated with clinically aggressive phenotypes, poor prognosis, and shorter survival (16 -21). Previously we and others have solved the x-ray crystal structure of the wild-type fascin (22)(23)(24). Although there are no internal homologous amino acid sequence repeats, the crystal structure of fascin consists of four structurally homologous tandem ␤-trefoil folds that form a two-lobed structure with pseudo 2-fold symmetry.…”

“…Jansen et al (24) employed structure-guided mutagenesis studies to reveal that fascin mutants K22E/K43E/R398E and R271E/K353E/K358E were defective in bundling actin filaments. In this report, we have used systematic mutagenesis studies and examined 100 fascin mutants.…”