Phosphomimic S3D Cofilin Binds Actin Filaments but does not Sever them

Cofilin-mediated remodeling of the actin cytoskeleton is critical for many cellular processes. Cofilinchanges the filamentstructure and renders them more compliant in bending and twisting. Filamentspartially decorated with cofilinsever more readily than bare or saturated ones, supporting a model where severing occurs preferentially at mechanical and topological boundaries between bare and cofilin-decorated segments. Phosphorylation of cofilinat serine 3 by LIM kinaseis a well-established mechanism for regulating cofilinactivity. Substitution of serine with aspartate at position three (S3D) is widely used for investigating the mechanism of cofilinphospho-regulation in cells and in biochemical studies with purified protein components. The S3D substitution or phosphorylation weakens cofilinbinding to actin filaments, and it is thought that subsequent reduction in cofilinoccupancy inhibits filamentsevering activity. Here, we show that S3D cofilinbinds actin filamentswith a lower affinity than wild-type (WT) cofilin, but with higher cooperativity. Because of its higher cooperativity, S3D cofilinwill form larger clusters along filamentsthan unmodified cofilinand thus have fewer boundaries between bare and decorated segments where severing occurs. S3D decoration weakly affects filamentbending and twisting dynamics, in contrast to WT cofilin. Weak actin filamentsevering is observed for S3D across a range of occupancies. Reduced boundary density and inability to alter filamentmechanics make S3D severing deficient.