Three distinct F-actin binding sites in the Dictyostelium discoideum 34 000 Dalton actin bundling protein

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 2 95 amino acids. However, the region(s) of the molecule that bind actin fila ments is tare) unknown. Studies of the cosedimentation of I-125-34 kDa prot ein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 mu m and 0.6 mu m, respectively. The Hill coefficient is larger for short filam ents that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sit es were identified using a synthetic peptide, protein truncations, and a no vel epitope library screening method. The ability to bind actin was assesse d by I-125- F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The thr ee actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. T he estimated K-app obtained by the disassembly of pyrene-labeled actin was 0.11 mu M and 2.7 mu M for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that m ay contribute to the positive cooperative formation of F-actin bundles.