Two distinct sites of interaction form the calponin: gelsolin complex and two calcium switches control its activity

Gelsolin and calponin are well characterized actin-binding proteins that form a tight gelsolin:calponin complex (GCC). We show here that the GCC is formed through two distinct interfaces. One of these is formed between 144-182 of calponin and 25-150 of gelsolin (G1). The second is a calcium-sensitive site centred on calponin's CH domain, and the C-terminal half of gelsolin (G4-6). The behaviour of this second interface is dependent on the presence of calcium and so it is possible that potential GCC-binding partners may be selected by calcium availability. Actin is one such GCC-binding partner and we show that a larger complex is formed with monomeric actin in calcium. The stoichiometry of this complex is determined to be 1 gelsolin/1 calponin/2 G-actins (GCA(2)). Both actin monomers bind the GCC through gelsolin. Both calponin and gelsolin are reported to play signaling roles in addition to their better-characterized actin-binding properties and it is possible that the GCC regulates both of these functions.     

The gelsolin:calponin complex nucleates actin filaments with distinct morphologies

Gelsolin and calponin are cytoskeletal and signalling proteins that form a tight 1:1 complex (GCC). We show that calponin within the GCC inhibits the rate of gelsolin mediated nucleation of actin polymerization. The actin-binding function of calponin is ablated within the GCC as the actin-binding site overlaps with one of the gelsolin binding sites. The structure of filaments that result from nucleation by GCC are different to those nucleated by gelsolin alone in that they are longer, loosely bundled and stain heterogeneously with phalloidin. GCC nucleated filaments appear contorted and wrap around each to form the loose bundles.     

Human plasma gelsolin binds to fibronectin

Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.     

Actin is a noncompetitive plasmin inhibitor.

Actin, one of the most abundant cellular proteins, circulates at micromolar concentrations in peripheral blood. Because actin released from dying cells may be trapped in fibrin clots that form at sites of tissue injury, we examined the effects of actin upon lysis of fibrin clots in vitro. Incorporation of native rabbit skeletal muscle actin into fibrin clots slowed their rates of lysis for periods of up to 24 h, an effect not seen when comparable concentrations of human IgG or bovine serum albumin were added instead. Actins isolated from a variety of sources inhibited plasmin's hydrolysis of the synthetic substrate S-2251 in a noncompetitive manner, with a Ki of a 0.6-3.1 microM. Inhibition was rapid, but covalent actin-plasmin complexes were not formed. Both epsilon-aminocaproic acid and tranexamic acid prevented actin's inhibition of plasmin, suggesting that accessible lysine residues of actin interact with the kringle (lysine-binding) regions of plasmin. Neither of the high-affinity actin-binding proteins of plasma (plasma gelsolin and vitamin D-binding protein) prevented actin from inhibiting plasmin. These findings suggest that actin released into the extracellular space following cell death may modulate plasmin action, and hence a number of plasmin-dependent biological responses, at sites of inflammation and tissue injury.     

Mutual effects of α-actinin,calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (α-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin α-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of α-actinin and calponin to actin bundles. Higher ability of calponin to depress α-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin–α-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with α-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that α-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Mutual effects of alpha-actinin, calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

Calponin binds G-actin and F-actin with similar affinity

Calponins are actin-binding proteins that are implicated in the regulation of actomyosin. Calponin binds filamentous actin (F-actin) through two distinct sites ABS1 and ABS2, with an affinity in the low micromolar range. We report that smooth muscle calponin binds monomeric actin with a similar affinity (K(d) of 0.15 microM). We show that the arrangement of binding is similar to that of F-actin by a number of criteria, most notably that the distance between Cys273 on calponin and Cys374 of actin is 29A when measured by fluorescent resonance energy transfer, the same distance as previously reported for F-actin.     

The CH-domain of calponin does not determine the modes of calponin binding to F-actin

Many actin-binding proteins have been observed to have a modular architecture. One of the most abundant modules is the calponin-homology (CH) domain, found as tandem repeats in proteins that cross-link actin filaments (such as fimbrin, spectrin and alpha-actinin) or link the actin cytoskeleton to intermediate filaments (such as plectin). In proteins such as the eponymous calponin, IQGAP1, and Scp1, a single CH-domain exists, but there has been some controversy over whether this domain binds to actin filaments. A previous three-dimensional reconstruction of the calponin-F-actin complex has led to the conclusion that the visualized portion of calponin bound to actin belongs to its amino-terminal homology (CH) domain. We show, using a calponin fragment lacking the CH-domain, that this domain is not bound to F-actin, and cannot be positioning calponin on F-actin as hypothesized. Further, using classification methods, we show a multiplicity in cooperative modes of binding of calponin to F-actin, similar to what has been observed for other actin-binding proteins such as tropomyosin and cofilin. Our results suggest that the form and function of the structurally conserved CH-domain found in many other actin-binding proteins have diverged. This has broad implications for inferring function from the presence of structurally conserved domains.     

Synthetic actin-binding domains reveal compositional constraints for function

The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.     

Immunological and biochemical studies on the relationship between two actin-binding proteins, phosphofructokinase and gelsolin

Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.     

Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin–actin interaction

Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1.     

Domain structure in actin-binding proteins: expression and functional characterization of truncated severin

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.     

Isolation and properties of two actin-binding domains in gelsolin

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.     

Dissecting the gelsolin-polyphosphoinositide interaction and engineering of a polyphosphoinositide-sensitive gelsolin C-terminal half protein

Gelsolin and other proteins in the villin/gelsolin family are regulated by polyphosphoinositides (PPIs), and manipulation of cellular PIP(2) levels alters the structure of the actin cytoskeleton coincident with the dissociation of gelsolin-actin complexes. This work explores the structure-function relationship of the gelsolin-PPI interaction. Circular dichroism experiments show that upon binding to PPIs, the PPI-sensitive N-terminal half of gelsolin undergoes significant secondary and tertiary structural changes that do not occur in the structurally homologous but PPI-insensitive C-terminal half. Secondary structure modeling algorithms predict an alpha-helical conformation for one of the gelsolin PPI-binding sites, P2, which differs from the conformation of P2 in the structure of gelsolin determined by X-ray crystallography, whereas structure prediction of the C-terminal homolog of P2 agrees well with the X-ray crystallography structure. Simulation of a change to helical conformation for P2 using molecular modeling indicates that such a structural transition will destabilize the F-actin-binding sites in domain 2. A hypothesis is proposed that PPIs initiate conformational changes at the PPI-binding site(s) that destabilize the protein structure, and subsequently disrupt the actin-binding sites. To further evaluate the role of P2 in the gelsolin-PPI interaction, a Ct mutant P2Ct is constructed by inserting P2 in place of its C-terminal homologous site. P2Ct interacts with actin in the same way as the wild-type protein. In contrast to Ct, however, P2Ct interacts strongly with PPIs, and its monomeric actin-binding activity becomes regulated by PPIs. It is concluded that the P2 site is sufficient for PPI-sensitivity in gelsolin. Furthermore, the P2 site in P2Ct and the actin-binding sites of Ct do not overlap, suggesting that PPIs regulate actin binding of P2Ct through induction of structural changes, rather than through direct competition.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.     

Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.     

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.     

The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex

Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1-G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca(2+) concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting gelsolin to bind to and sever actin filaments. How Ca(2+) is able to activate gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of gelsolin bound to monomeric actin at 3.0 A resolution. Two classes of Ca(2+)-binding site are evident on gelsolin: type 1 sites share coordination of Ca(2+) with actin, while type 2 sites are wholly contained within gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six domains of gelsolin suggests that this site exists in each of the domains. In total, gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of gelsolin by releasing the tail latch that locks calcium-free gelsolin in a conformation unable to bind actin.