title: 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 β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-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 β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 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.
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Jansen
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Collins
authors.forename: Changsong
authors.initials: C
authors.lastname: Yang
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Rebowski
authors.forename: Tatyana
authors.initials: T
authors.lastname: Svitkina
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D008840:Microfilament Proteins; C020324:fascin; D011493:Protein Kinase C