Purification and characterization of actobindin, a new actin monomer-binding protein from Acanthamoeba castellanii

Actobindin is a new actin-binding protein isolated from Acanthamoeba castellanii. It is composed of two possibly identical polypeptide chains of approximately 13,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and with isoelectric points of 5.9. In the native state, actobindin appears to be a dimer of about 25,000 daltons by sedimentation equilibrium analysis. It contains no tryptophan and probably no tyrosine. Actobindin reduces the concentration of F-actin at steady state and inhibits the rate of filament elongation to extents consistent with the formation of a 1:1 actobindin-G-actin complex in a reaction with a KD of about 5 microM. The available data do not eliminate the possibility of other stoichiometries for the complex, but they are not consistent with any significant interaction between actobindin and F-actin. Despite the similarities between the effects of actobindin and Acanthamoeba profilin on the polymerization of Acanthamoeba actin, the two proteins are quite distinct with different native and subunit molecular weights, different isoelectric points, and different amino acid compositions. Also, unlike profilin, actobindin binds as well to rabbit skeletal muscle G-actin and to pyrenyl-labeled G-actin as it does to unmodified Acanthamoeba G-actin.     

C-terminal variations in beta-thymosin family members specify functional differences in actin-binding properties

Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.     

beta-Thymosins, small acidic peptides with multiple functions

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.     

The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif

Actobindin is an 88-amino acid polypeptide, containing two almost identical repeated domains of 33 and 34 residues. Depending on the molar ratios in which they are mixed, actobindin binds either one or two actin molecules. We cross-linked actobindin and actin in the 1:1 complex, using the zero-length cross-linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. The cross-linked peptides were purified after consecutive CNBr cleavage and trypsin and Staphylococcus protease V8 digestions, and the cross-linked side chains were identified by amino acid sequencing. Isopeptide linkages were formed between residues Glu-100 of actin and Lys-16 of actobindin. In addition, we found a connection between one or more of the acidic residues 1,2, or 3 of actin and Lys-16 and Lys-52 of actobindin. The cross-linked regions in actobindin contain Leu-Lys-His-Ala-Glu-Thr motifs, similar to sequences observed in several other actin-binding proteins.     

The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.     

Effects of Nucleotide and End-Dependent Actin Conformations on Polymerization

The regulation of actin is key for controlled cellular function. Filaments are regulated by actin-binding proteins, but the nucleotide state of actin is also an important factor. From extended molecular dynamics simulations, we find that both nucleotide states of the actin monomer have significantly less twist than their crystal structures and that the ATP monomer is flatter than the ADP form. We also find that the filament’s pointed end is flatter than the remainder of the filament and has a conformation distinct from G-actin, meaning that incoming monomers would need to undergo isomerization that would weaken the affinity and slow polymerization. Conversely, the barbed end of the filament takes on a conformation nearly identical to the ATP monomer, enhancing ATP G-actin’s ability to polymerize as compared with ADP G-actin. The thermodynamic penalty imposed by differences in isomerization for the ATP and ADP growth at the barbed end exactly matches experimental results.     

Inhibition of an early stage of actin polymerization by actobindin

Actobindin, a 25,000-dalton dimeric protein purified from Acanthamoeba castellanii was previously shown to form a 1:1 molar complex with both Acanthamoeba and rabbit muscle G-actin with KD values of about 5 and 7 microM, respectively, and not to interact with F-actin (Lambooy, P. K., and Korn, E. D. (1986) J. Biol. Chem. 261, 17150-17155). We now find that actobindin is a much more potent inhibitor of the early phases of polymerization of both Acanthamoeba and muscle G-actin than can be accounted for by its binding to G-actin. Actobindin inhibits the polymerization of both G-ATP-actin and G-ADP-actin, and has little, if any, effect on the rate of ATP hydrolysis that accompanies polymerization of G-ATP-actin. The kinetics of actin polymerization in the presence of actobindin are qualitatively consistent with the postulation that actobindin binds reversibly to and inhibits the elongation of an intermediate between G-actin and F-actin, perhaps a small oligomer(s) or a species in equilibrium with such an intermediate. This hypothesis implies the, at least transient, existence of an actin species with properties different from those of monomers and filaments. Actobindin may, then, provide a useful experimental tool for investigating the still relatively obscure early steps in actin polymerization. Irrespective of its mechanism of action, actobindin might serve in situ to reduce the rate of actin polymerization de novo while having relatively little effect on the rates of elongation of existing filaments or from actobindin-resistant nucleating sites.     

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.     

The interaction of monomeric actin with two binding sites on Acanthamoeba actobindin

Actobindin was previously shown to be an 88-residue polypeptide (Mr 9761) with an internal tandem repeat of 33-34 amino acids. Sedimentation equilibrium experiments have confirmed this Mr for native actobindin. Pyreneglyoxal-labeled actobindin had a similar Mr by sedimentation equilibrium analysis and bound to actin in a manner qualitatively similar to unmodified actobindin as determined by gel electrophoretic analysis of covalently cross-linked products. The stoichiometry of the actin-actobindin interaction was determined from the change in apparent Mr of pyrene-glyoxal-labeled actobindin in the presence of actin, as determined by scanning the ultracentrifuge cell at a wavelength that detected only the labeled protein. These data were consistent with the formation of a complex containing two actin and one actobindin molecules. The overall KD describing the binding of the first actin to either of the two sites on actobindin was 3.3 microM. The binding constant for the second actin suggested either negative cooperativity or inequality of the two actin-binding sites. Similar binding constants were obtained by analysis of the fluorescence enhancement that occurred when actobindin bound to actin labeled with either pyrene iodoacetamide or 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole. Cross-linking experiments with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide qualitatively agreed with predictions made from a two-binding site model. Additionally, both the fluorescence and cross-linking experiments suggested that the interaction of the two actin molecules may contribute to the stability of the heterotrimeric complex.     

How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.     

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.     

The covalent structure of Acanthamoeba actobindin

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.     

Spire and Cordon-bleu: multifunctional regulators of actin dynamics

WASP-homology 2 (WH2) domains, which were first identified in the WASP/Scar (suppressor of cAMP receptor)/WAVE (WASP-family verprolin homologous protein) family of proteins, are multifunctional regulators of actin assembly. Two recently discovered actin-binding proteins, Spire and Cordon-bleu (Cobl), which have roles in axis patterning in developmental processes, use repeats of WH2 domains to generate a large repertoire of novel regulatory activities, including G-actin sequestration, actin-filament nucleation, filament severing and barbed-end dynamics regulation. We describe how these multiple functions selectively operate in a cellular context to control the dynamics of the actin cytoskeleton. In vivo, Spire and Cobl can synergize with other actin regulators. As an example, we outline potential methods to gain insight into the functional basis for reported genetic interactions among Spire, profilin and formin.     

Phosphorylation by actin kinase of the pointed end domain on the actin molecule.

Fragmin from plasmodium of Physarum polycephalum binds G-actin and severs F-actin in the presence of Ca2+ over 10(-6) M. The fragmin-actin complex consisting of fragmin and G-actin nucleates actin polymerization and caps the barbed (fast growing) end of F-actin, regardless of the concentrations of Ca2+, and the actin filaments are shortened. Actin kinase purified from plasmodium abolishes the nucleation and capping activities of the complex by phosphorylating actin of the fragmin-actin complex (Furuhashi, K., and Hatano, S. (1990) J. Cell. Biol. 111, 1081-1087). This inactivation of the complex leads to production of long actin filaments. We obtained evidence that Physarum actin is phosphorylated by actin kinase at Thr-201, and probably at Thr-202 and/or Thr-203, with 1 mol of phosphate distributed among them. This finding raises the possibility that the site of phosphorylation, Thr-201 to Thr-203, is positioned on the pointed (slow growing) end domain of the actin molecule, because growth of actin filaments from the fragmin-actin complex occurs only from the pointed end. These observations are consistent with a model of the three-dimensional structure of G-actin. Inactivation of the fragmen-actin complex may follow phosphorylation of the pointed end domain of actin.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeats

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.     

UNC-87 is an actin-bundling protein

The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and alpha-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.     

Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids.

Chicken muscle CapZ, a member of the capping protein family of actin-binding proteins, binds to the barbed end of actin filaments and nucleates actin polymerization. No regulation of the capping protein family has been described. We report that micelles of phosphatidylinositol 4,5-bisphosphate (PIP2) bind to CapZ and completely inhibit its ability to affect actin polymerization as measured by several independent assays. Higher concentrations of other anionic phospholipids also completely inhibit the activity of CapZ. Neutral phospholipids have no effect. Mixed vesicles of PIP2 with phosphatidylcholine or phosphatidylethanolamine also inhibit CapZ, but addition of Triton X-100 both prevents and reverses PIP2's inhibition of CapZ.     

Analysis of the function of Spire in actin assembly and its synergy with formin and profilin

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.