Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function

Abstract

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter 'actin crossovers' ( approximately 75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.

Figure 1

Cofilin and ADF change the twist of rabbit muscle F-actin. Electron micrographs of negatively stained F-actin (a) and filaments decorated with cofilin (b) and ADF (c) at pH 6.5. Both cofilin and ADF induce a 25% reduction in crossover length. White bars indicate crossover positions. Bar, 0.1 μm.

Figure 2

Cofilin changes the twist of platelet F-actin. (a) Portion of a computationally straightened F-actin filament; (b) portion of a computationally straightened actin filament decorated with cofilin. The defocus of both micrographs shown was 1.5 μm. Decorated filaments are ∼120–130 Å diam with accentuated ‘crossovers' relative to control actin. (c) Computed diffraction pattern calculated from the actin filament shown in (a). The length of the filament used to calculate this pattern was 1.44 μm. (d) Computed diffraction pattern calculated from the cofilin-decorated actin filament shown in (b). The length of the filament used to calculate this pattern was 1.41 μm. Prominent layerlines in each diffraction pattern are labeled with values of n and l. The axial position of layerline n = 2, l = 4 in the F-actin diffraction pattern is 1/366 Å⁻¹ and that of n = 2, l = 2 in the cofilin/F-actin diffraction pattern is 1/272 Å⁻¹.

Figure 3

Cooperativity of cofilin binding to rabbit muscle F-actin at pH 6.5. 6 μM F-actin was cosedimented with cofilin (0–35 μM). Supernatant and pellets were analyzed by SDS-PAGE and concentrations of cofilin and actin estimated by densitometry. Bound cofilin is expressed as a molar ratio to actin subunits. Nonlinear least-squares fitting shows a maximum binding at 1.16 cofilin/actin and a Hill constant of 6.4.

Figure 4

Cooperativity of cofilin binding to rabbit muscle F-actin shown by electron cryomicroscopy. (a) Micrograph of frozen- hydrated filaments decorated with cofilin at subsaturating conditions. (b and c) Micrographs of partially decorated filaments: the top region of each filament is thinner, bare F-actin, whereas the lower region is decorated with cofilin. (d and e) Computed diffraction patterns calculated from the bare F-actin and decorated F-actin images in a. (f and g) Computed diffraction patterns from the undecorated and decorated halves of filament b. (h and i) Computed diffraction patterns from the undecorated and decorated halves of filament c. In this case the decorated region of the filament is only about four crossovers long. This results in a broadening of the layerline data i. Arrowheads indicate the positions of equivalent (J₂) layerlines.

Figure 5

Reconstructions of F-actin and cofilin/actin filaments demonstrating the change in crossover length. (a) Platelet F-actin exhibiting 54/25 symmetry, equivalent to 2.160 subunits per turn. (b) Cofilin/F-actin exhibiting 20/9 symmetry (2.222 subunits per turn). Arrows mark the crossover length. There are 40 actin subunits in each filament.

Figure 6

Identification of the cofilin-binding site by difference mapping. (a) Platelet actin reconstruction brought to the twist of the cofilin/actin filament (2.222 subunits per turn). The subdomains of actin and the phalloidin-binding site (yellow asterisk) are indicated. (b) Cofilin/actin reconstruction at the same orientation as in (a). (c) Positive difference density (copper) calculated by subtracting a from b. The contour level chosen encloses 100% (nominal) molecular volume. (d) Effects of contouring the cofilin/actin reconstruction using high thresholds. The actin reconstruction is shown as a transparent surface (silver) and the cofilin-decorated filament (gold) at two different molecular volumes (40 and 10%) to emphasize the strongest densities in the map.

Figure 7

Atomic modeling of cofilin binding to F-actin. (a) Stereo images showing the placement of the difference density calculated from the cofilin/F-actin structure on the model filament. Magenta asterisks show the positions of His 40, 87 with 88, and 101. (b) Stereo images showing the model placement of the destrin coordinates on the filament based on the cofilin/F-actin reconstruction. This model can be generated by starting with the proposed placement of destrin on G-actin (Hatanaka et al., 1997) and applying the following matrix and vector translation:

	0.8515	0.5072	0.1331
	−0.3293	0.7147	−0.6170
	−0.4081	0.4815	0.7756
	−1.7062	−12.3144	−12.5306

The NH₂ terminus of destrin is oriented at ∼2 o'clock in this figure (near His 40 of the lower actin monomer).