Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

The actin depolymerizing factor (ADF)/cofilin family of proteins interact w ith actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of f ilaments, thereby increasing treadmilling or depolymerization. Using site-d irected mutagenesis we characterized the two actin-binding sites on human c ofilin. One target site was chosen because we previously show?ed that the v illin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essentia l for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix alre ady implicated in actin binding. Binding through the alpha-helix is primari ly to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Usi ng the mutation analogous to the phosphorylated form of cofilin (S3D), we s how that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces b oth the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, foll owed by interaction with the adjacent subunit through the "helix" site at t he opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsi ble for the enhanced rate of actin subunit release from filaments. (C) 2000 Academic Press.