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 severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy 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 cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification ( substitution with Asp or phosphorylation) "undocks" and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit 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 filament mechanical properties and promote severing.

The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments

“…Estimates of the length over which cofilininduced conformational changes and cooperative binding interactions propagate along actin vary, ranging from N = 1-2 up to N > 100 subunits (3,5,(12)(13)(14)(15)(16)(17)(18)(19)(20). Equilibrium (3,6,12,21) and transient kinetic (12,14) binding data are well described by models invoking positive cooperativity between nearest neighbors (N = 1-2).…”

“…Samples with a cofilin binding density (v) of ~0.5 cofilin per actin were used for cryo-EM sample preparation. Cofilin binding to unlabeled actin was measured by cosedimentation (19).…”

“…Immediately before polymerization, Ca-actin monomers were converted to Mg-actin with addition of EGTA and MgCl 2 and equilibrated on ice for 5 min. (34 (3,12,19). Samples with a cofilin binding density (v) of ~0.5 cofilin per actin were used for cryo-EM sample preparation.…”

“…Rabbit skeletal muscle actin and recombinant human non-muscle cofilin-1 proteins were purified as described (19). Actin was labeled with pyrene (N-(1-pyrene)Iodoacetamide, ThermoFisher Scientific, cat.…”

Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly

“…A prevalent factor is cofilin-1 Ser3 phosphorylation by LIM kinases (Arber et al, 1998;Kanellos and Frame, 2016). This phosphorylation strongly and persistently inhibits cofilin-1 as it reduces its affinity for actin filaments and slows down subsequent severing (Elam et al, 2017). Additionally, cofilin-1 activity can be restrained by tropomyosins (Tpm) (Brayford et al, 2016;Christensen et al, 2017;Gateva et al, 2017;Jansen and Goode, 2019).…”