Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.     

Crystal structure of polymerization-competent actin

All actin crystal structures reported to date represent actin complexed or chemically modified with molecules that prevent its polymerization. Actin cleaved with ECP32 protease at a single site between Gly42 and Val43 is virtually non-polymerizable in the Ca-ATP bound form but remains polymerization-competent in the Mg-bound form. Here, a crystal structure of the true uncomplexed ECP32-cleaved actin (ECP-actin) solved to 1.9 A resolution is reported. In contrast to the much more open conformation of the ECP-actin's nucleotide binding cleft in solution, the crystal structure of uncomplexed ECP-actin contains actin in a typical closed conformation similar to the complexed actin structures. This unambiguously demonstrates that the overall structure of monomeric actin is not significantly affected by a multitude of actin-binding proteins and toxins. The invariance of actin crystal structures suggests that the salt and precipitants necessary for crystallization stabilize actin in only one of its possible conformations. The asymmetric unit cell contains a new type of antiparallel actin dimer that may correspond to the "lower dimer" implicated in F-actin nucleation and branching. In addition, symmetry-related actin-actin contacts form a head to tail dimer that is strikingly similar to the longitudinal dimer predicted by the Holmes F-actin model, including a rotation of the monomers relative to each other not observed previously in actin crystal structures.     

An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells

The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells.     

Structure of the alpha-actinin rod: molecular basis for cross-linking of actin filaments.

We have determined the crystal structure of the two central repeats in the alpha-actinin rod at 2.5 A resolution. The repeats are connected by a helical linker and form a symmetric, antiparallel dimer in which the repeats are aligned rather than staggered. Using this structure, which reveals the structural principle that governs the architecture of alpha-actinin, we have devised a plausible model of the entire alpha-actinin rod. The electrostatic properties explain how the two alpha-actinin subunits assemble in an antiparallel fashion, placing the actin-binding sites at both ends of the rod. This molecular architecture results in a protein that is able to form cross-links between actin filaments.     

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.     

Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution

The structure of a dimer of the Escherichia coli catabolite gene activator protein has been refined at 2.5 A resolution to a crystallographic R-factor of 20.7% starting with coordinates fitted to the map at 2.9 A resolution. The two subunits are in different conformations and each contains one bound molecule of the allosteric activator, cyclic AMP. The amino-terminal domain is linked to the smaller carboxy-terminal domain by a nine-residue hinge region that exists in different conformations in the two subunits, giving rise to approximately a 30 degree rotation between the positions of the small domains relative to the larger domains. The amino-terminal domain contains an antiparallel beta-roll structure in which the interstrand hydrogen bonding is well-determined. The beta-roll can be described as a long antiparallel beta-ribbon that folds into a right-handed supercoil and forms part of the cyclic AMP binding site. Each cyclic AMP molecule is in an anti conformation and has ionic and hydrogen bond interactions with both subunits.     

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.     

Exogenous nucleation sites fail to induce detectable polymerization of actin in living cells

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.     

Cross-linked dimers with nucleating activity in actin prepared from muscle acetone powder

A covalently linked actin dimer is identified in solutions of actin prepared from an acetone powder from skeletal muscle. This actin dimer acts as an actin nucleating factor (ANF), decreasing the half-time for spontaneous actin polymerization. ANF reacts with antibodies to both the N- and C-terminal portions of actin on Western blots and migrates during reduced polyacrylamide gel electrophoresis like actin cross-linked with N, N'-p-phenylenebismaleimide. The origin of the cross-linked dimer appears to be related to the presence of carbonyl groups in purified actin. A large number of carbonyls (approximately 0.3/actin) are introduced into actin during the prolonged treatment with acetone in the preparation of the muscle acetone powder from which actin is extracted. Actin extracted from acetone powder prepared by a single acetone wash and actin prepared from bovine spleen, which is not washed with acetone, both contain fewer carbonyl groups (approximately 0.05 carbonyl/actin). ANF forms spontaneously in solutions of polymer actin containing 0.3 carbonyl/actin. We speculate that a reaction between a carbonyl on one actin polymer subunit and a lysine on a neighboring subunit is responsible for ANF formation. The presence of cross-linked actin dimers in commonly used skeletal muscle actin preparations could certainly affect studies of actin polymerization and, particularly, studies of the nucleation reaction. The physiological relevance of ANF is not clear, but given the large cellular concentration of actin, similar reactions yielding ANF could occur in vivo when increased levels of reactive oxygen species are present.     

Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer

Actin filament nucleation, polymerization, and branching are crucial steps in many forms of cell motility, cell shape, and intracellular organelle movements in a wide range of organisms. Previous biochemical data suggests that an anti-parallel actin dimer can incorporate itself into growing filamentous actin (F-actin) and has a role in branching. Furthermore, it is a widespread belief that nucleation is spawned from an actin trimer complex. Here we present the structures of actin dimers and trimers in two tetragonal crystal systems P4(3)2(1)2 and P4(3). Both crystal systems formed by an induced condensation transformation of a previously reported orthorhombic crystal system P2(1)2(1)2(1). Comparison between the three crystal systems demonstrates the dynamics and flexibility of actin-actin interactions. The dimer and trimer actin rearrangements observed between the three crystal systems may provide insight to in vivo actin-actin interactions that occur during the nucleation, polymerization, and branching of F-actin.     

Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture

Formin proteins participate in a wide range of cytoskeletal processes in all eukaryotes. The defining feature of formins is a highly conserved approximately 400 residue region, the Formin Homology-2 (FH2) domain, which has recently been found to nucleate actin filaments. Here we report crystal structures of the S. cerevesiae Bni1p FH2 domain. The mostly alpha-helical FH2 domain forms a unique "tethered dimer" in which two elongated actin binding heads are tied together at either end by an unusual lasso and linker structure. Biochemical and crystallographic observations indicate that the dimer is stable but flexible, with flexibility between the two halves of the dimer conferred by the linker segments. Although each half of the dimer is competent to interact with filament ends, the intact dimer is required for actin nucleation and processive capping. The tethered dimer architecture may allow formins to stair-step on the barbed end of an elongating nascent filament.     

Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin beta(4). Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.     

Structure of an F-actin trimer disrupted by gelsolin and implications for the mechanism of severing

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.     

Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation

Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This stimulated nucleation activity is closely correlated with actin polymerization observed in vivo in its kinetics, developmental regulation, and cytochalasin D sensitivity. Actin polymerization is coordinate with pseudopod extension in synchronized populations of cells and is correlated with the accumulation of F actin in pseudopods. The stimulated actin nucleation activity is present in low-speed pellets from Triton lysates (cytoskeletons) within 3 s of stimulation and is stable compared with the nucleation activity of whole cell lysates. Low-speed supernatants contain a reversible inhibitor of the actin nucleation activity that is itself regulated by chemotactic stimulation. Neither activity requires Ca2+ and both are fully expressed in 10 mM EGTA. Fractions containing the inhibitor do not sever actin filaments but do inhibit actin polymerization that is seeded by fragments of purified F actin. These results indicate that chemotactic stimulation of Dictyostelium discoideum generates both an actin-nucleating activity and an actin-polymerization inhibitor, and suggest that the parallel regulation of these two activities leads to the transient phases of actin polymerization observed in vivo. The different compartmentation of these two activities may account for polarized pseudopod extension in gradients of chemoattractant.     

Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).     

Crystal structure of the alpha-actinin rod reveals an extensive torsional twist

BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. RESULTS: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.     

Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

The Rickettsia ～1800-amino-acid autotransporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments of Sca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.     

Control of actin polymerization in live and permeabilized fibroblasts

We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.     

Preparation,purification and properties of a crosslinked trimer of G-actin

Phenylenebismaleimide has been used to form crosslinks between actin monomers [Knight, P. and Offer, G. (1978) Biochem. J. $\text{175}$, 1023–1032]. We have purified a trimer of actin monomers as well as a dimer and a mixture of higher molecular weight oligomers. The trimer is much more effective than the dimer in enhancing the rate of polymerization while higher oligomers do not appear to be any more effective than the trimer. A lag in the polymerization process, as measured fluorescence enhancement of trace pyrene-actin, still occurs in the presence of trimers serving as the nuclei, suggesting that the mechanism for polymerization is more complex than nucleation followed by elongation.     

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.