The Crystal Structure of Plasma Gelsolin: Implications for Actin Severing, Capping, and Nucleation

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N -phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.Arguably the most regulated cytoskeletal protein, filamentous actin (F-actin) is critical for cellular motility and mechanical strength, protein sorting and secretion, signal transduction, and cell division. An atomic resolution understanding of the structure of F-actin is a tantalizing goal. The tendency of monomeric actin to form polymers of varying lengths has prevented crystallization and atomic resolution structural analysis of F-actin. To date, all but one of the atomic resolution structures of actin are of the monomer bound to proteins that prevent polymerization, such as DNaseI (2), GS-1 (3), profilin (4, 5), and, most recently, vitamin D-binding protein (6, 7). A structure of uncomplexed monomeric actin labeled with the dye tetramethylrhodamine (8) reveals that actin-binding proteins did not alter the structure of the actin monomer significantly. The structure of an antiparallel actin dimer has been determined (9), but how this structure relates to helical F-actin is unclear.A model describing F-actin as a helical polymer has been proposed based on fitting the crystal structure of monomeric actin into x-ray fiber diffraction data from oriented actin gels (1). Schutt and co-workers (4, 5, 10) have constructed a topologically different helical model for F-actin by twisting the ribbon-like assembly of actin molecules found in profilin-actin crystals. Transitioning between twisted and untwisted forms of this ribbon was proposed to be a basis for contractile force generation (11-13). To date, the ribbon actin assembly has been observed only in the crystals of profilin-actin heterodimers (4, 5, 10).A key component to the dynamic as...

“…Ca 2ϩ Is Inhibited by Phalloidin-Analogous to full-length gelsolin (38,39), the severing activity of GS-1 is Ca 2ϩ -dependent (Fig. 8C, red).…”

Section: Gs-1-mediated Depolymerization Of F-actin In the Presence Ofmentioning

confidence: 99%

Structure of an F-actin Trimer Disrupted by Gelsolin and Implications for the Mechanism of Severing

Dawson¹,

Sablin²,

Spudich³

et al. 2003

“…Gelsolin is a calciumsensitive actin-binding, severing, and nucleating protein that is composed of six similar domains (G1-6) (71). Although the six domains are similar in both sequence and structure (28), it is known that whereas G1 and G4 bind a similar site on actin (72,73), G2 binds F-actin alone. G2 is thought to bind actin in the region of the outer surfaces of subdomains 1 and 2 (62).…”

Section: Involvement Of Actin Subdomain 1 In the Interaction Withmentioning

confidence: 99%

“…26) and to the gelsolin-villin family of ABPs because the gelsolin fold (27,28) is similar to that of the ADF-cofilin fold (29).…”

mentioning

confidence: 99%

The Identification of a Second Cofilin Binding Site on Actin Suggests a Novel, Intercalated Arrangement of F-actin Binding

Renoult

Ternent²,

Maciver³

et al. 1999

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44). We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047-1055).Many motile processes in cells require cyclic polymerization and depolymerization of actin filaments. In cell locomotion for example, actin is polymerized at the leading edge of the cell and is recycled by depolymerizing toward the cell center. The rate constants of pure actin have been established (1), and it is clear that a discrepancy exists between these known rates and those calculated from filament turnover in cells (2). A host of actinbinding proteins are known that dramatically alter the behavior of actin in vitro, and of these, the cofilins have been suggested to have the correct properties to increase filament turnover in cells (3). This view has been confirmed by studies with living Saccharomyces (4) and Dictyostelium (5) and by Listeria motility assays (6, 7).The cofilins are a group of low molecular mass (15-21 kDa), actin-binding proteins that depolymerize actin filaments (8).This group includes vertebrate cofilin (9) and ADF 1 (10), twinstar from Drosophila (11), depactin from echinoderms (12), ADFs from plants (13), Unc-60 from nematode (14), cofilins from Saccharomyces (15, 16) and Dictyostelium (17), and actophorin from Acanthamoeba castellanii (18).The mechanism by which cofilin depolymerizes actin filament has been contentious. Soon after the discovery of the first member of the family (10), several authors suggested that depolymerization occurred through severing (9, 18). Evidence for a severing mechanism later came from videomicroscopy (19,20), but this was later challenged (6) as it was shown (6, 21) that cofilins increased the off-rate from the pointed end of the filament. However, the two opinions are not necessarily exclusive (22-24), and a similar mechanism has been proposed for both events (23). We report the identification of a cof...

“…These isoforms exhibit a compact globular structure under Ca 2ϩ -free conditions at the physiological pH and attain an open functionally active structure in the presence of 1 mM Ca 2ϩ or at lower pH (2)(3)(4). Radiolytic footprinting and small angle x-ray scattering (SAXS) 4 studies have elucidated a three-stage opening of the gelsolin molecule upon sequential binding of free Ca 2ϩ .…”

mentioning

confidence: 99%

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Peddada¹,

Sagar²,

Rathore³

et al. 2013

Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma gelsolin. Results: N-terminal fragment 30 -161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca 2ϩ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca 2ϩ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.