Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

Abstract Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severingby the regulatory protein cofilin. Phosphorylation of vertebrate cofilinat Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilinphosphorylation in cells and in vitro. The S3D substitution weakens cofilinbinding to filaments, and it is presumed that subsequent reduction in cofilinoccupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilinindeed binds filamentswith lower affinity, but also with a higher cooperativity than wild-type cofilin, and seversactin weakly across a broad range of occupancies. We found that three factors contribute to the severingdeficiency of S3D cofilin. First, the high cooperativity of S3D cofilingenerates fewer boundaries between bare and decorated actin segments where severingoccurs preferentially. Second, S3D cofilinonly weakly alters filamentbending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severingat boundaries. Third, Ser-3 modification (i.e. substitution with Asp or phosphorylation) “undocks” and repositions the cofilinN terminus away from the filamentaxis, which compromises S3D cofilin's ability to weaken longitudinal filamentsubunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filamentmechanical properties and promote severing.