Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

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' (~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.     

The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site

Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.     

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.     

Toxoplasma gondii Actin Depolymerizing Factor Acts Primarily to Sequester G-actin

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the related actin depolymerizing factor (ADF) and cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin-binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the ADF/cofilin (AC) family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer-sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.     

Antagonistic effects of cofilin, beryllium fluoride complex, and phalloidin on subdomain 2 and nucleotide-binding cleft in F-actin

Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound epsilon-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of epsilon-ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.     

The second ADF/cofilin actin-binding site exists in F-actin, the cofilin-G-actin complex, but not in G-actin.

ADF/cofilins are actin binding proteins that bind actin close to both the N- and C-termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112-125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol. 138, 771-781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G-actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G-actin by FRET induced by cofilin, to reveal the second cofilin-binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation.     

Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface

ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR-actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments.     

Energetics and kinetics of cooperative cofilin-actin filament interactions

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.7 thermodynamically bound counterions. Counterion dissociation contributes approximately 40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments

Previously, we have shown that the V-ATPase holoenzyme as well as the V1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V1 subunits B and C (Vitavska, O., Wieczorek, H., and Merzendorfer,H. (2003) J. Biol. Chem. 278, 18499-18505). Since the recombinant subunit C not only enhances actin binding of the V1 complex but also can bind separately to F-actin, we analyzed the interaction of recombinant subunit C with actin. We demonstrate that it binds not only to F-actin but also to monomeric G-actin. With dissociation constants of approximately 50 nm, the interaction exhibits a high affinity, and no difference could be observed between binding to ATP-G-actin or ADP-G-actin, respectively. Unlike other proteins such as members of the ADF/cofilin family, which also bind to G- as well as to F-actin, subunit C does not destabilize actin filaments. On the contrary, under conditions where the disassembly of F-actin into G-actin usually occurred, subunit C stabilized F-actin. In addition, it increased the initial rate of actin polymerization in a concentration-dependent manner and was shown to cross-link actin filaments to bundles of varying thickness. Apparently bundling is enabled by the existence of at least two actin-binding sites present in the N- and in the C-terminal halves of subunits C, respectively. Since subunit C has the possibility to dimerize or even to oligomerize, spacing between actin filaments could be variable in size.     

Structural conservation between the actin monomer-binding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin

Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins. In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin. To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis. This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin. Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin. In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin. This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins. Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins.     

肌动蛋白解聚因子/cofilin功能的研究进展

肌动蛋白解聚因子（actin depolymerizing factor,ADF）/cofilin家族是一类肌动蛋白结合蛋白,它们通过切断肌动蛋白纤丝并结合到肌动蛋白单体上,在重塑肌动蛋白骨架中发挥重要作用。就ADF/cofilin家族的结构特点、调控肌动蛋白动力学的机制及其功能的最新研究进展做一简要综述,并指出了目前在ADF/cofilin功能研究方面的不足和尚需解决的问题。     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.     

Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.     

A structural basis for the pH-dependence of cofilin. F-actin interactions.

A marked pH-dependent interaction with F-actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin-binding proteins. ADF/cofilins tend to bind to F-actin with a ratio of 1 : 1 at pH values around 6.5, and to G-actin at pH 8.0. We have investigated the mechanism for the pH-sensitivity. We found no evidence for pH-dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G-actin. None of the actin-derived, cofilin-binding peptides that we had previously identified [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899] bound cofilin in a pH-sensitive manner. However, we have detected a conformational change in region 75-105 in the actin subdomain 1 by the use of a peptide-directed antibody. A pH-dependent conformational change has also been detected spectroscopically in a similar peptide (84-103) on binding to cofilin. These results are consistent with a model in which pH-dependent motion of subdomain 1 relative to subdomain 2 (through region 75-105) of actin reveals a second cofilin binding site on actin (centered around region 112-125) that allows ADF/cofilin association with the actin filament. This motion requires salt in addition to low pH.     

Functional Surfaces on the Actin-binding Protein Coronin Revealed by Systematic Mutagenesis

Coronin is a conserved actin-binding protein that co-functions with ADF/cofilin and Arp2/3 complex to govern cellular actin dynamics. Despite emerging roles for coronin in a range of physiological processes and disease states, a detailed understanding of the molecular interactions of coronin with actin and other binding partners has been lacking. Here, we performed a systematic mutational analysis of surfaces on the yeast coronin β-propeller domain, which binds to F-actin and is conserved in all coronin family members. We generated 21 mutant alleles and analyzed their biochemical effects on actin binding and ADF/cofilin activity. Conserved actin-binding residues mapped to a discrete ridge stretching across one side of the β-propeller. Mutants defective in actin binding showed loss of synergy with ADF/cofilin in severing filaments, diminished localization to actin structures in vivo, and loss of coronin overexpression growth defects. In addition, one allele showed normal actin binding but impaired functional interactions with ADF/cofilin. Another allele showed normal actin binding but failed to cause coronin overexpression defects. Together, these results indicate that actin binding is critical for many of the biochemical and cellular functions of coronin and that the β-propeller domain mediates additional functional interactions with ADF/cofilin and possibly other ligands. Conservation of the actin-binding surfaces across distant species and in all three major classes of coronin isoforms suggests that the nature of the coronin-actin association may be similar in other family members.