Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca²⁺-regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.     

Binding of myotrophin/V-1 to actin-capping protein: implications for how capping protein binds to the filament barbed end

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a Kd of 10-50 nm. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP beta subunit was important for binding V-1, with lesser contributions from the alpha subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1.CP complex had no capping activity. Two loops of V-1, which extend out from the alpha-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the beta tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the alpha1 and alpha2 isoforms as well as the mutant lacking the alpha tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile beta-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.     

Electron microscopic visualization of the filament binding mode of actin-binding proteins

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.     

Conformational dynamics of capping protein and interaction partners: simulation studies

Capping protein (CP) is important for the regulation of actin polymerization. CP binds to the barbed end of the actin filament and prevents actin polymerization. This interaction is modulated through competitive binding by regulatory proteins such as myotrophin (V-1) and the capping protein interacting (CPI) motif from CARMIL. The binding site of myotrophin overlaps with the region of CP that binds to the barbed end of actin filament, whereas CPI binds at a distant site. The binding of CPI to the myotrophin-CP complex dissociates myotrophin from CP. Detailed multicopy molecular dynamics simulations suggest that the binding of CPI shifts the conformational equilibria of CP away from states that favor myotrophin binding. This shift is underpinned by allosteric effects where CPI inhibits CP through suppression of flexibility and disruption of concerted motions that appear to mediate myotrophin binding. Accompanying these effects are changes in electrostatic interactions, notably those involving residue K142β, which appears to play a critical role in regulating flexibility. In addition, accessibility of the site on CP for binding the key hydrophobic residue W8 of myotrophin is modulated by CPI. These results provide insights into the modulation of CP by CPI and myotrophin and indicate the mechanism by which CPI drives the dissociation of the myotrophin-CP complex.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

A structural basis for regulation of actin polymerization by pectenotoxins

(PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification of their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin, which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts, allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure-activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

High microfilament concentration results in barbed-end ADP caps.

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

In vivo importance of actin nucleotide exchange catalyzed by profilin

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.     

The Interaction of Vinculin with Actin

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.     

Two biochemically distinct and tissue-specific twinfilin isoforms are generated from the mouse Twf2 gene by alternative promoter usage

Twf (twinfilin) is an evolutionarily conserved regulator of actin dynamics composed of two ADF-H (actin-depolymerizing factor homology) domains. Twf binds actin monomers and heterodimeric capping protein with high affinity. Previous studies have demonstrated that mammals express two Twf isoforms, Twf1 and Twf2, of which at least Twf1 also regulates cytoskeletal dynamics by capping actin filament barbed-ends. In the present study, we show that alternative promoter usage of the mouse Twf2 gene generates two isoforms, which differ from each other only at their very N-terminal region. Of these isoforms, Twf2a is predominantly expressed in non-muscle tissues, whereas expression of Twf2b is restricted to heart and skeletal muscle. Both proteins bind actin monomers and capping protein, as well as efficiently capping actin filament barbed-ends. However, the N-terminal ADF-H domain of Twf2b interacts with ADP-G-actin with a 5-fold higher affinity than with ATP-G-actin, whereas the corresponding domain of Twf2a binds ADP-G-actin and ATP-G-actin with equal affinities. Taken together, these results show that, like Twf1, mouse Twf2 is a filament barbed-end capping protein, and that two tissue-specific and biochemically distinct isoforms are generated from the Twf2 gene through alternative promoter usage.     

Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.     

Mechanism for CARMIL protein inhibition of heterodimeric actin-capping protein

Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ～30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.