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Phosphomimetic S3D cofilin binds but only weakly severs actin filaments - PubMed

  • ️Sun Jan 01 2017

. 2017 Dec 1;292(48):19565-19579.

doi: 10.1074/jbc.M117.808378. Epub 2017 Sep 22.

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Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

W Austin Elam et al. J Biol Chem. 2017.

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 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.

Keywords: actin; cofilin; cooperativity; molecular dynamics; spectroscopy.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.

S3D cofilin binds actin filaments with lower affinity but higher cooperativity than WT cofilin. Shown are equilibrium binding titrations of WT (black circles) or S3D (white circles) cofilin and pyrene-labeled actin filaments. The solid lines through the data represent the best fits to Equations 2 and 4.

Figure 2.
Figure 2.

A higher S3D binding cooperativity reduces the number of boundaries between bare and cofilin-decorated segments. The boundary density calculated using Equation 5 is plotted against binding density for ligands that bind non-cooperatively (ω = 1, solid line) and cooperatively with ω = 5 (dashed line), 50 (dotted line), or 500 (dashed-dotted line). Note that the boundary density is symmetric and peaks at 50% occupancy (ν = 0.5).

Figure 3.
Figure 3.

S3D binding affinity but not cooperativity is linked to cation release. A, binding titrations of S3D (from left to right) in 10 (squares), 25 (circles), 50 (upright triangles), 75 (diamonds), or 100 m

m

KCl (rightward triangles). B, binding titrations of S3D (from left to right) in 0.5 (squares), 2 (circles), or 5 m

m

MgCl2 (triangles). The smooth lines though data in A and B are fits to Equations 2 and 4. C and D, the KCl and MgCl2 concentration dependence of the intrinsic S3D cofilin binding affinity (Kd) (C) and binding cooperativity (ω) (D) obtained from the fits to Equations 2 and 4 in A and B. KCl and MgCl2 dependence of Kd in C yields slopes of −1.9 ± 0.6 and −1.4 ± 0.2, respectively, corresponding to dissociation of ∼2.3 K+ or ∼0.9 Mg2+ with S3D binding (see Ref. for details on calculations). These values compare with the number of ions released with WT cofilin binding (Table 2) (27). In D, S3D cofilin binding cooperativity depends weakly on the solution salt concentration, similarly to WT cofilin (27).

Figure 4.
Figure 4.

S3D cofilin severs weakly and inclusion of competitors enhances severing activity. A, normalized equilibrium average lengths (Lavg) of Alexa-labeled actin filaments at various WT (black squares) or S3D (white circles) binding densities, n ≥ 200 per condition. Solid black lines though WT and S3D cofilin binding data represent fits of the data to a discrete, site-specific severing model (Equation 1) (14) with fixed cooperativity values of ω = 5 and 47, respectively. The top horizontal dotted line with the value Lavg = 1 denotes average length of bare actin filaments. The dashed black line shows a simulation of average filament lengths (Equation 1) with WT cofilin apparent severing rate constants but with S3D cooperativity (ω = 47), demonstrating that the increased cooperativity alone cannot account for S3D severing deficiency. Error bars, S.E. B, binding competition of phalloidin (black circles) or non-muscle tropomyosin Tpm3.1 (white circles) with S3D for pyrene-labeled actin filaments. Smooth lines though data are fits to the Hill equation and are strictly for visualization purposes. C, equilibrium mean lengths (open squares in boxes) of Alexa-labeled actin filaments that are either bare, half-decorated with S3D cofilin, or partially decorated with S3D cofilin and a competitor (Tm or Ph). Equilibrium distribution of filament lengths (n ≥ 200) is indicated by a 25 or 75% marker (horizontal line) connected by vertical dashed lines. The marker × indicates percentile 1 or 90, whereas the floating marker (heavy horizontal line) represents the maximum or minimum of filament length.

Figure 5.
Figure 5.

S3D decoration weakly affects actin filament torsional dynamics. Shown are phosphorescence anisotropy decays for ErIA-labeled actin, WT-decorated (ν ∼0.9), and S3D-decorated (ν ∼0.9) fitted to a double exponential (smooth lines through data). Fit residuals are 1–2% of signal, and the fitting parameters are listed in Table 5. The inset shows data in log scale to reveal differences at early time scales. A model-dependent analysis (12) of the anisotropy decays was also performed to calculate the filament intersubunit torsional constant α and torsional rigidity C (Table 4).

Figure 6.
Figure 6.

Wild-type cofilin– and S3D cofilin–decorated actin filaments adopt similar structures. A and B, electron cryomicroscopy structure of S3D cofilin–decorated (A) or WT cofilin–decorated (B) actin filaments overlaid with an atomic WT cofilin–decorated actin filament model (Protein Data Bank code 3J0S) (10). Cofilin is colored blue, and actin subunits are colored green or yellow. C and D, close-up view of a single cofilin subunit (WT, S3D) with the N terminus of cofilin indicated with a red arrow.

Figure 7.
Figure 7.

Serine 3 modification repositions the cofilin N terminus away from the filament. Top, isolated single cofilactin interface from MD simulation of a cofilactin filament (11 actin filament subunits with 11 bound cofilin molecules). Actin is colored red, whereas cofilin is colored blue with N-terminal residues 1–4 shown as space-filling models. Bottom, time course of the distance in average position of the four cofilin N-terminal residues from the center of actin subdomain 1, as observed in MD simulations. The WT cofilin N terminus remains docked to the adjacent actin subunit, but either mutation of Ser-3 (S3D) or phosphorylation (S3Ph) compromises this docking, as indicated by the increased distance between the cofilin N terminus and actin subdomain 1. See also

supplemental Movies 1 and 2

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References

    1. Bamburg J. (1999) Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu. Rev. Cell. Dev. Biol. 15, 185–230 - PubMed
    1. Pollard T. D., and Borisy G. G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 - PubMed
    1. Kanellos G., and Frame M. C. (2016) Cellular functions of the ADF/cofilin family at a glance. J. Cell Sci. 129, 3211–3218 - PubMed
    1. De La Cruz E. (2005) Cofilin binding to muscle and non-muscle actin filaments: isoform-dependent cooperative interactions. J. Mol. Biol. 346, 557–564 - PubMed
    1. De La Cruz E. (2009) How cofilin severs an actin filament. Biophys. Rev. 1, 51–59 - PMC - PubMed

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