Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

W. Austin Elam, Wenxiang Cao, Hyeran Kang, Andrew Huehn, Glen M. Hocky, Ewa Prochniewicz, Anthony C. Schramm, Karina Negrón, Jean Garcia, Teresa T. Bonello, Peter W. Gunning, David D. Thomas, Gregory A. Voth, Charles V. Sindelar, Enrique M. De La Cruz

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


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 in vitro. 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 (i.e. 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.

Original languageEnglish (US)
Pages (from-to)19565-19579
Number of pages15
JournalJournal of Biological Chemistry
Issue number48
StatePublished - Dec 1 2017

Bibliographical note

Funding Information:
2 Supported by an American Heart Association Postdoctoral Fellowship. Pres-ent address: C4 Therapeutics, 675 W. Kendall St., Cambridge, MA 02142.

Funding Information:
6 Supported by the National Science Foundation, Grant DBI-1156585, and by the Raymond and Beverly Sackler Institute for Biological, Physical and Engi-neering Sciences.

Funding Information:
4 Supported by a Ruth L. Kirschstein National Research Service Award (NIGMS, National Institutes of Health, Grant F32 GM11345-01).

Funding Information:
8 Supported by NHMRC Grant APP1004188 and the Kids Cancer Network.

Funding Information:
This work was supported by National Institutes of Health R01 Grants GM097348 (to E. M. D. L. C.), GM110533001 (to C. V. S.), and AR032961 (to D. D. T.); American Cancer Society Grant IRG5801255 (to C. V. S.); and the Department of Defense Army Research Office through MURI Grant W911NF1410403 (on which G. A. V. and E. M. D. L. C. are co-investigators). The authors declare that they have no conflicts of interest with the con-tents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Insti-tutes of Health.

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