FIGURE 3.

Identification of two major actin-binding sites in β-trefoil domains 1 and 3. A, two views of the surface of the fascin structure rotated by ∼180° and colored by residue conservation (conservation decreases from blue to red as indicated by the bottom bar). For reference, small domain-colored diagrams in the same orientation are shown alongside. The alignment of fascin sequences used to determine the conservation score of each residue is shown in supplemental Fig. 5. Conservation scores were calculated and displayed with the programs ConSurf (19) and PyMOL (Schrödinger, LLC), respectively. Note that the two largest patches of conserved residues occur in β-trefoil domains 1 and 3. The patch in β-trefoil-1 comprises residue Ser³⁹. The antimetastatic drug macroketone (5) also binds at the edge of this patch (shown, although not part of this structure). In the current structure, molecules of polyethylene glycol and glycerol bind in pockets within the two conserved patches and could guide the design of anticancer drugs. Residues mutated in this study are labeled. B, actin binding and bundling assays with fascin mutants 1 and 3 targeting the conserved patches in β-trefoil domains 1 and 3 (conducted as in Fig. 2A). S, supernatant; P, pellet. C, transmission electron micrographs of negatively stained actin filaments alone and in the presence fascin mutants 1 and 3 (conducted as in Fig. 2B). These mutants have almost completely lost the ability to bundle actin but can bind to the sides of individual actin filaments (arrowheads). D, quantitative analysis of the bundles formed by mutants 1 and 3 (conducted as in Fig. 2C). Error bars, S.D.