An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability

Abstract

The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.

The actin cytoskeleton plays an essential role in diverse cellular processes ranging from motility to division. A key control point in the cycle of actin filament (F actin) assembly is the rate-limiting nucleation step, which can be accelerated in a regulated manner by the action of nucleating factors. One of the major actin-nucleating factors in cells is the Arp2/3 complex, a protein complex that consists of seven subunits including the actin-related proteins (Arp) Arp2 and Arp3 and the additional Arp2/3 complex (ARPC) polypeptides ARPC1–ARPC5. The Arp2/3 complex has been conserved during the evolution of most eukaryotic cells and plays an important functional role in cell migration, endocytosis, phagocytosis, and pathogen infection (1).

On its own the Arp2/3 complex is inactive, but it is activated to polymerize actin by binding to proteins called nucleation-promoting factors (NPFs) (1) as well as to ATP (2, 3). Moreover, the nucleating activity of the Arp2/3 complex is stimulated by binding to F actin (4, 5), a phenomenon that results in autocatalytic actin assembly (6). Once activated, the complex nucleates the polymerization of daughter filaments that emerge from the sides of mother filaments in a stereotypical Y-branch orientation with an approximate branch angle of 70° (7–9). Such Arp2/3-containing branched structures have been observed in the actin network within lamellipodia at the leading edge of motile cells (10). This branched filament geometry is proposed to be particularly suited for harnessing actin polymerization to generate motile force (11).

Atomic-resolution structures of the Arp2/3 complex with and without bound nucleotide and inhibitors have been determined (12–15). Moreover, structural models of the Y-branch junction have been constructed using electron microscopy (16, 17), culminating in a 2.6-nm resolution 3D model derived from docking crystal structures of Arp2/3 complex and actin into a reconstruction from electron tomography (18). In this model, Arp2 and Arp3 interact with the pointed end of the daughter filament, and all seven subunits contact the mother filament. ARPC2 and ARPC4 comprise the major mother-filament-binding interface, consistent with earlier data from chemical cross-linking (19), Arp2/3 complex reconstitution (20), and antibody-inhibition experiments (21). However, despite advances in our understanding of Y-branch structure, the functional importance of Arp2/3 complex residues implicated in mother-filament binding has not been extensively tested apart from an analysis of ARPC2 (arc35) mutants in Saccharomyces cerevisiae, which demonstrated an important role for ARPC2 residues in Arp2/3 complex nucleating activity in vitro and in growth and endocytosis in vivo (22).

In this study we used molecular dynamics and protein-protein docking simulations to generate an independent model of the interaction between the ARPC2 and ARPC4 subunits of the Arp2/3 complex and the side of an actin filament. Using information from this and previous models, we tested the role of amino acid residues on the exposed surfaces of ARPC2 and ARPC4 by examining the biochemical properties of mutant Arp2/3 complexes. Using this approach we defined key residues that play a specific and critical role in F-actin binding, actin nucleation, and Y-branch stability.

Results

Protein-Protein Docking Simulations Yield an Independent Structural Model of ARPC2/ARPC4 Bound to F Actin.

Numerous lines of evidence suggest that the ARPC2 and ARPC4 subunits of the Arp2/3 complex constitute the primary F-actin side-binding interface (19–21). To generate an independent structural model of the interaction between these proteins, we performed protein-protein docking simulations using the crystal structures of an ARPC2/ARPC4 heterodimer (12) and an actin filament consisting of eight monomers corresponding to the Holmes F-actin model (23). To capture the conformational flexibility of the two docking partners, we used Nanoscale Molecular Dynamics (NAMD) (24) to perform a 100-ns molecular dynamics simulation of the full Arp2/3 complex and a 30-ns simulation of the actin filament. Structures were extracted every 10 ns for the ARPC2/ARPC4 heterodimer and every 5 ns for F actin, resulting in 10 and 6 structures, respectively. For each combination of structures, we performed 100 docking simulations using Rosetta-Dock software (25, 26), resulting in a total of 6,000 docked structures.

Using the best-scoring model from this initial protein-protein docking run as a starting point, we carried out a perturbation run in which the two docking partners were subjected to an additional 10,000 rounds of random translation/rotation and redocking. When we examined the resulting energy landscape using the final best-scoring structure as a reference, we observed a funnel of docking scores converging on the lowest-scoring structure at 0 Å rmsd (Fig. S1). Score funnels such as this one were found to be a strong indicator of the robustness of the docking model, as the low-scoring model in such funnels often corresponded with atomic-level accuracy to the structure determined experimentally by x-ray crystallography in the Critical Assessment of Predicted Interactions (CAPRI) protein-protein docking experiment (27). Thus, the model with the lowest docking score from the perturbation run, shown in Fig. 1A and B and Movie S1, represents a robust computationally derived model of the ARPC2/ARPC4-F-actin-binding interaction.

Fig. 1.

Protein-protein docking simulations identify a putative mother filament binding site on the Arp2/3 complex. (A) Surface rendering of the structures of an ARPC2/ARPC4 heterodimer and a portion of the actin filament, with residues predicted to form salt bridges highlighted in red (acidic) and blue (basic), and the surface predicted to be within 4 Å of the other binding partner in yellow, based on molecular dynamics and protein-protein docking simulations. Each structure is rotated 90° away from the other to show the binding interfaces. (B) Structures of the whole Arp2/3 complex with the predicted mother filament binding interface highlighted in yellow. (C) Structure of the inactive Arp2/3 complex (12) docked onto a mother filament in the orientation dictated by the position of ARPC2/ARPC4 in the top-scoring model from docking simulations.

Strikingly, when we overlaid our model of the ARPC2/ARPC4-F-actin interaction on the one derived from electron microscopy (Movie S1), we found they were very similar, with a C_α rmsd of only 5.9 Å. The similarity between these models provides independent support for the results from electron microscopy studies. Moreover, the robustness of the protein-protein docking model is highlighted by the fact that it was derived independently of information from electron microscopy.

Encouraged by the similarity of our protein-protein docking model with models from electron microscopy, we identified the interaction surface on ARPC2/ARPC4 that is predicted to lie within 5 Å of F actin based on the protein-protein docking analysis. This interface resides primarily on ARPC4 (residues M1, A3, T4, L5, R55, N56, E57, K58, E59, K77, Q78, A79, D80, E81, E83, D143, K144, S147, K150, L151, S152, N154, A155, R158, I159, E162, E163, and K166) but extends along ARPC2 (residues D159, R160, E187, R189, R190, A191, H193, F228, Y261, and R265) at the junction between the two proteins. The surface on F actin primarily resides on subdomains 1 and 2 of a single monomer (residues E2, D3, E4, T5, Q41, M44, Q49, K50, D51, S52, K68, N78, D80, D81, K84, H87, H88, Y91, N92, R95, A97, E99, E100, H101, F127, and N128 in rabbit muscle α-actin). Most of the residues on ARPC2/ARPC4 predicted by protein-protein docking to lie within 5 Å of actin are also predicted to lie within 5 Å of actin by electron microscopy (18) (Table S1). Interestingly, some of these residues were also proposed to be involved in mother-filament binding by a homology modeling study that identified highly conserved amino acids within the Arp2/3 complex (28) (Fig. 2A and Table S1). Together, the protein-protein docking, electron microscopy, and homology modeling approaches made complementary and testable predictions about the potential importance of specific Arp2/3 complex surface residues in its nucleation and actin-binding activities.

Fig. 2.

Residues on ARPC2 and ARPC4 identified by molecular docking and homology modeling lie on a surface that is critical for Arp2/3 complex activity. (A) Surface rendering showing the location of amino acid residues selected for mutagenesis (opaque and color coded) based on molecular docking (blue), homology modeling (green), or both (magenta). (B) Surface rendering showing the correlation between the location of mutations on the surface of ARPC2/ARPC4 and the severity of the actin nucleation defects. The surface that is predicted to be within 4 Å of actin by protein-protein docking is opaque and yellow, and the side chains of mutated amino acids are shown in space filling representation and are color coded as follows: orange, severely defective; purple, moderately defective; green, unaffected.

Charged Surface Residues on ARPC2 and ARPC4 Are Critical for Arp2/3 Complex Activity.

We set out to test the functional importance of charged surface residues on ARPC2 and ARPC4 by mutating clusters of these residues to alanine. We generated a total of 11 mutant complexes that contained substitutions in 21 different amino acid residues (Table S2 and Figs. S2 and S3). Each of the mutant complexes was expressed in insect cells using the baculovirus expression system and purified to homogeneity. None of the mutations had an effect on the stability or assembly of the complex, as each purified complex was obtained with similar yield and had the appropriate subunit stoichiometry.

We assessed each purified complex for its ability to promote actin polymerization using the pyrene-actin assembly assay (Fig. 3). Because Arp2/3-mediated actin nucleation is autocatalytic, we expected that mutations affecting F-actin binding would decrease activity. Of the 11 mutants tested, 8 exhibited reduced activity and 3 exhibited near-wild-type activity (Fig. 3A and Table S2). To quantify these defects, we used the polymerization data to calculate the concentration of actin filament barbed ends generated by each mutant complex as a function of Arp2/3 concentration (Fig. 3B). Mutations causing reduced activity diminished the production of barbed ends from 2-fold (2DR, 4RE) to 12-fold (4DKK) (Table S2). These data indicate that numerous charged residues on the surface of ARPC2 and ARPC4 play a critical role in Arp2/3 complex function.

Fig. 3.

Mutations in charged surface residues on ARPC2 and ARPC4 cause a range of actin nucleation defects. (A) Pyrene-actin polymerization assays with wild-type (WT) and mutant Arp2/3 complexes. Reactions contain 3 µM actin, either alone (actin) or with 20 nM WT or the indicated mutant complexes (see Table S2 for abbreviations) and 200 nM GST-WASP-WCA. Mutant complexes with similar activity levels are grouped together and colored according to the scheme in Fig. 2B. (B) Concentration of barbed ends generated by WT and mutant Arp2/3 complexes as a function of Arp2/3 concentration.

We next examined the relationship between the location of each mutation on the surface of ARPC2/ARPC4 and the severity of the biochemical defect. Strikingly, when the mutations were mapped onto the surface, there was a clear spatial clustering by activity level (Fig. 2B). Mutations that caused moderate to severe biochemical defects clustered on the predicted binding interface with F actin, and the least severe mutations scattered at the periphery or outside the predicted interface. Notably, 12 of the 14 mutations that caused moderate to severe defects are predicted to be within 5 Å of the mother filament, whereas only 1 of 7 mutations that caused little defect are within close proximity to actin (Table S1). Thus the locations of mutations that influence actin polymerization support the electron microscopy and protein-protein docking models for the interaction of ARPC2 and ARPC4 with the mother actin filament.

A Surface on ARPC2/ARPC4 Is Critical for F-Actin Binding and Y-Branch Stability.

We hypothesized that the mutations in residues in ARPC2 and ARPC4 that compromised actin polymerization activity did so by reducing the affinity for F actin, but not by perturbing the interaction of the Arp2/3 complex with other activators such as ATP or NPFs. To test this, we first used wild-type and mutant Arp2/3-FRET complexes (containing ARPC1 tagged with YFP and ARPC3 tagged with CFP) (29) as conformational sensors of ATP and NPF binding. No defects in ATP- or NPF-induced conformational changes were observed for mutants with the most (4DKK) and least (4RED) severe nucleation defects (Fig. 4A), suggesting that deficiencies in nucleating activity are not due to a failure to bind nucleotide or NPF. Next, we used cosedimentation at a range of F-actin concentrations to measure binding to actin filaments (Fig. 4B). The affinity of the severely defective 4DKK mutant for F actin (K_d = 7.8 ± 3.7 μM; mean ± standard error of the mean) was 6 times lower than wild type (K_d = 1.3 ± 0.6 μM). This strongly suggests that the reduced polymerizing activity of 4DKK, and likely the other mutants, is due to a reduced affinity for actin filaments.

Fig. 4.

Mutant Arp2/3 complexes undergo expected conformational changes on activator binding, but fail to bind F actin with high affinity or form long-lasting branches. (A) Box and whiskers plot of normalized FRET/CFP ratio of WT, 4DKK, and 4RED Arp2/3-FRET complexes in the presence of Mg²⁺, Mg-ATP, or Mg-ATP and GST-WASP-CA. The middle line of each box indicates the median, and the top and bottom lines represent the third and first quartiles (n = 6). Whiskers indicate maximum and minimum measurements. (B) Percentage of WT (black) or 4DKK (orange) Arp2/3 complex found in the pellet fraction after high-speed centrifugation over a range of actin concentrations. Dissociation constants (K_ds) calculated from the resulting curves are indicated. Data are the mean ± SEM (n = 6). (C) Images of branches formed by WT and the 4DKK complexes. Scale bar 2 µm. (D) Graph of the fraction of the initial branching frequency (normalized such that the branch frequency at t = 0 is 1) vs. time after initiating nucleation/branching for WT (black) and 4DKK (orange). Data are the mean ± SEM (n = 3).

Finally, we compared the activity of the wild-type complex and the 4DKK mutant with regard to the formation and subsequent stability of Y branches. After controlling for the 12-fold difference in actin-nucleating activity, wild type and 4DKK formed a similar percentage of branched filaments (10 nM wild type formed 15 ± 3% branched filaments (mean ± SEM); 120 nM 4DKK formed 13 ± 1% branched filaments), and branch morphology was similar between wild type and mutant (Fig. 4C). This suggests that the lower affinity of the 4DKK mutant results in lower nucleation and Y-branching activity, but nevertheless its nucleating and Y-branching activities remain tightly coupled as has been shown previously for wild-type Arp2/3 complex (4–6). In addition to measuring Y-branch formation, we also measured Y-branch stability by initiating nucleation/branching reactions, fixing branches with rhodamine-phalloidin at various times post initiation, and quantifying branch frequency as a function of time (Fig. 4D). Wild-type branches dissociated with a t_1/2 (half life) of 28 min, similar to previous reports (30). In striking contrast, branches formed by the 4DKK mutant dissociated with a much shorter t_1/2 of < 10 min. This indicates that residues within the ARPC2/ARPC4 heterodimer that affect the affinity of the Arp2/3 complex for F actin are crucial for Y-branch stability.

Discussion

By employing a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between the ARPC2 and ARPC4 subunits of the Arp2/3 complex and the side of the mother filament. Our model corresponds remarkably well to previous models of the Arp2/3 complex in the Y branch derived from electron microscopy (16–18). Together these models provide candidate contact sites between the binding partners. By mutating residues within the predicted actin-binding surface on ARPC2/ARPC4, we defined sites on this surface that are required for high-affinity binding to F actin, efficient actin nucleation and Y branching, and stability of Y branches.

Our results suggest that the F-actin-binding region on ARPC2/ARPC4 occupies much of the exposed surface of ARPC4 and extends onto ARPC2 near the interface between the two proteins. The surface is very similar to that obtained by fitting a crystal structure of the Arp2/3 complex into the density of Y-branch junctions observed by electron tomography (18) and encompasses some of the evolutionarily conserved residues that were suggested to be involved in F-actin binding by homology modeling (28). Mutating residues that span this surface caused moderate to severe defects in actin polymerization, supporting the notion that this surface is critical for Arp2/3 complex activity.

When we compared the relative success of protein-protein docking (this study), electron microscopy (18), and homology modeling (28) in predicting functionally critical residues on this surface of ARPC2/ARPC4, we found that protein-protein docking and electron microscopy were comparably successful, and both were more successful than homology modeling. Of the 14 residues for which mutation caused a moderate/severe phenotype, protein-protein docking predicted that 12 were at the F-actin-binding interface, compared with 13 for electron microscopy, and 9 for homology modeling. Conversely, of the 7 residues for which mutations caused little phenotype, protein-protein docking predicted that 6 were not at the interface, compared with 4 for electron microscopy, and 3 for homology modeling. Thus all of these approaches have predictive power, and each arrives at a complementary solution. The combination of these methods provides a larger measure of confidence and makes a broader set of predictions that can be tested experimentally.

It is important to note that the results of our mutagenesis experiments agree with a previous analysis of the phenotypes caused by mutating evolutionarily conserved and solvent-exposed residues in S. cerevisiae ARPC2 (22). In particular, mutating several residues that are conserved between yeast and human ARPC2 caused correspondingly mild (yeast arc35-107; human 2EE; E204A), moderate (yeast arc35-104; human 2DR; D159A R160A), and severe (yeast arc35-106; human 2ER, 2EER; E187A R190A) defects in actin nucleation. Our results demonstrate that the functional roles of these residues are conserved across species and extend this analysis to examine the role of adjacent residues in ARPC4.

Detailed examination of the biochemical activity of a severely defective mutant in ARPC4 (4DKK) suggests that this surface of ARPC4 (and by extension the adjacent surface on ARPC2) is crucial for actin nucleation and high-affinity binding to F actin, consistent with the observations that F-actin binding plays a key role in activating the complex (4–6). Although the precise mechanism of Arp2/3 complex activation by F actin remains unclear, mathematical simulations suggest that an activation reaction occurs after actin binding and that this is the rate-limiting step leading to branch formation (31). In addition to their role in promoting high-affinity actin binding, residues on the surface of ARPC2/ARPC4 may also participate in this reaction. Once activation occurs, our data suggest that Y branches form with normal geometry even when these residues are mutated. However, our results also indicate that residues in ARPC2/ARPC4 are crucial for maintaining the stability of the Y branch, as the 4DKK mutant undergoes much more rapid branch dissociation than the wild-type Arp2/3 complex. Together these data suggest that residues on this surface of ARPC2/ARPC4 are crucial for F-actin binding and that the affinity for F actin independently affects both Arp2/3 complex activation and Y-branch stabilization.

In addition to the key role played by ARPC2 and ARPC4, results from electron microscopy suggest that each of the remaining five subunits makes contact with the mother filament (18), indicating that other interactions are also likely to be relevant in the intact complex. When we added the structures of the remaining subunits (12) to the model of ARPC2/ARPC4 docked on the mother filament to generate a model of the Arp2/3 complex bound to F actin (Fig. 1C), we found that ARPC5 is very close to the filament (4 Å), whereas other subunits are more distant (Arp2, 23 Å; ARPC1, 11 Å; ARPC3, 22 Å). However, the crystal structure we used for subunit placement is presumed to be an inactive conformation, and significant conformational changes are thought to occur during activation (12) that bring other subunits into closer proximity with F actin (18). Insight into the potential impetus for these conformational changes comes from our observation that, when all of the subunits are added to the docking model, there is a steric clash between Arp3 (subdomain 2) and F actin. This suggests that binding of the inactive Arp2/3 complex to F actin may result in a repositioning of Arp3, leading to the large-scale rearrangements in the complex that have been suggested to accompany the transition between the inactive conformation and the structure in the branch junction (12, 18).

Our results also suggest that the ARPC2/ARPC4 binding interface on F actin primarily encompasses a surface on subdomains 1 and 2 of a single actin monomer, similar to the binding surface predicted by electron microscopy (18). Importantly, our protein-protein docking model likely represents an encounter complex, as structural rearrangements in the mother filament beyond what was sampled in our simulations are seen by electron microscopy (18). Interestingly, one of the residues in actin (R95) that was predicted by our analysis to form a salt bridge with ARPC2/ARPC4 was also implicated in binding to tropomyosin based on chemical modification studies (32), consistent with the observation from electron microscopy that the binding sites on F actin for ARPC2/ARPC4 and tropomyosin overlap (18). This offers an explanation for the findings that tropomyosin inhibits actin nucleation and branching by the Arp2/3 complex in vitro (33) and that tropomyosin and Arp2/3 complex localize to distinct zones of the actin network in cellular lamellipodia (34).

The success of modeling a structure of the ARPC2 and ARPC4 subunits bound to F actin highlights the utility of combining molecular dynamics simulations, protein-protein docking simulations, and biochemical analyses to model protein-protein interactions. Future modeling efforts will require information from additional electron microscopy and x-ray crystallography studies aimed at determining both lower and higher resolution structures of Arp2/3 complex in various conformations and bound to its activators. The structural predictions from this and future studies will be invaluable for guiding investigation into both the mechanism by which F-actin binding promotes Arp2/3 complex activation and the functional importance of Y branching by Arp2/3 complex in cells.

Experimental Procedures

Molecular Dynamics and Docking Simulations.

The crystal coordinates for Arp2/3 complex (12) and ADP-actin (35) were obtained from the Protein Data Bank (PDB ID codes 1K8K and 1J6Z, respectively). The Arp2/3 complex structure was missing a considerable portion of Arp2, and smaller portions of the other six subunits. The missing portion of Arp2 was rebuilt and the nucleotides were added to Arp2 and Arp3 using Protein Local Optimization Program (PLOP) (36). The structure of the actin filament was constructed by replacing monomers in the Holmes model (23) with the ADP-actin crystal structure. Using the molecular dynamics package NAMD (37), simulations of Arp2/3 complex and actin filaments were performed using the CHARMM27 force field, an NpT ensemble at 1 atm pressure, a temperature of 300 K, a 10-Å cutoff for van der Waals interactions with a switching distance of 8.5 Å, and Particle Mesh Ewald for long-range electrostatics. Covalent bonds with hydrogens were held rigid allowing us to take 2-fs time steps. Following equilibration runs of 10–20 ns, the production F-actin simulation was 30 ns and the Arp2/3 complex simulation was 100 ns long. Structures were extracted every 5 ns for F actin and every 10 ns for the ARPC2/ARPC4 complex, resulting in 6 and 10 structures, respectively. Analysis of the rmsd and root mean square fluctuations of each simulation indicated that we were achieving reasonable sampling of the backbone and surface loops of each complex, and the inclusion of such flexibility was helpful in identifying the best-bound complex.

Using each combination of F actin and ARPC2/ARPC4 structures, we performed 100 docking runs using Rosetta-Dock (25), resulting in a total of 6,000 predicted complexes. Because of the multiple binding sites available to the ARPC2/ARPC4 complex on the actin filament, cluster analysis of the docking results yielded limited information. Therefore, we chose instead to base further analysis on the docking scores.

Starting with the best-scoring docked complex from the initial set of runs, we performed an additional 10,000 perturbation runs using the default dock_pert parameters in Rosetta-Dock. When we plotted the docking score vs. the rmsd, using the best structure from these perturbation runs as the reference, we saw the emergence of a funnel-shaped energy landscape, reminiscent of what has been observed in control dockings with native complexes (25–27). The best-scoring structure from this perturbation analysis was used for all subsequent work.

Baculovirus Strain Construction.

Baculovirus strains expressing untagged recombinant human Arp2/3 subunits, as well as strains expressing ARPC1-Flag-His₆, ARPC3-CFP, and ARPC1-YFP were generated as described previously (20, 29). Point mutations in ARPC2 and ARPC4 were made using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer’s protocol. The DNA sequence of each construct was verified, and then baculovirus strains were prepared according to the procedures supplied with the Bac-to-Bac system (Invitrogen).

Protein Expression and Purification.

Recombinant Arp2/3 complexes were expressed by infecting Hi5 cells with baculovirus strains expressing all Arp2/3 subunits including ARPC1-Flag-His₆, as described previously (29). Arp2/3 complexes were purified by Ni-NTA chromatography (QIAGEN) followed by cation exchange chromatography on HiTrap SP (GE Biosciences) for most complexes, or by anion exchange chromatography on HiTrap Q (GE Biosciences) for those complexes containing ARPC1-YFP. Ion exchange chromatography was followed by gel filtration (Superdex 200, GE Biosciences) into gel filtration buffer [20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.0, 100 mM KCl, 2 mM MgCl₂, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 0.2 mM ATP, and 10% glycerol]. When purifying complexes for use in FRET experiments, ATP, EGTA, EDTA, and MgCl₂ were omitted from gel filtration buffer (FRET gel filtration buffer) and from buffers used in other chromatography steps.

GST fusions of the Wiskott-Aldrich syndrome protein (WASP) WASP homology 2, connector, and acidic region (GST-WASP-WCA, residues 422–502 of human WASP) and the connector and acidic region (GST-WASP-CA, residues 441–502) were expressed in Escherichia coli BL21 (DE3) as described previously (38). These proteins were purified by glutathione affinity chromatography (glutathione sepharose 4B, GE Biosciences) and then by gel filtration chromatography (Superdex 200, GE Biosciences) into FRET gel filtration buffer. Protein was frozen in liquid nitrogen and stored at -80 °C.

Arp2/3 Complex Activity Assays.

Rabbit skeletal muscle actin (39) and pyrene-labeled actin (40) were prepared as described elsewhere. Pyrene-actin polymerization assays were performed and barbed end concentrations were calculated as described previously (20) with the following modifications. Pyrene-actin and unlabeled actin were mixed in G buffer (5 mM Tris pH 8.0, 0.2 mM CaCl₂, 0.2 mM ATP, 0.2 mM DTT) to generate a 3-µM G-actin solution with 7% pyrene-actin. A 14-µL gel filtration buffer or Arp2/3 complex, NPF, or other protein factor was mixed with 6 µL 10× initiation buffer (20 mM MgCl₂, 10 mM EGTA, 5 mM ATP), which was then mixed with a 40-µL G-actin mix to initiate polymerization. Polymerization reactions were allowed to reach steady state, and curves were normalized for differences in steady state fluorescence.

Actin copelleting assays were performed as described previously using 50-nM Arp2/3 complexes and the indicated concentrations of F actin (20). Curve fitting and K_d determination was performed using Prism software (GraphPad Software).

Debranching assays were performed essentially as described previously (30). Briefly, pyrene-actin assembly assays were performed as described above with wild-type (5 or 10 nM) or DKK mutant Arp2/3 complex (60 or 120 nM) and 200 nM GST-WASP-WCA. Samples were extracted at the indicated time points, and filaments were immediately stabilized by addition of rhodamine phalloidin (Invitrogen Molecular Probes). The first time point (t = 0) was taken when actin polymerization reached steady state. Rhodamine-phalloidin-stabilized filaments were applied to poly(L-lysine)coated cover slips and imaged using an Olympus BX51 microscope equipped with a 60× objective and a Hamamatsu Orca-ER camera. Images were captured in TIFF format using μManager software, and the frequency of branching was quantified by manual counting using Metamorph software (Molecular Devices). Images were prepared for presentation by adjusting brightness and contrast using Adobe Photoshop. Curve-fitting and Y-branch t_1/2 calculations were performed using Prism software (GraphPad Software).

FRET Experiments.

FRET experiments were carried out as described previously (29). Briefly, CFP and YFP tagged rArp2/3 complex (60 nM final concentration) was mixed with FRET gel filtration buffer (20 mM MOPS pH 7.0, 100 mM KCl, 0.5 mM DTT, 10% glycerol) or GST-WASP-WCA in a total of 9 µL. This was combined with a 48-µL FRET buffer (50 mM Tris pH 8.0, 112 mM KCl, 0.1 mM DTT) and 3-µL 20× MgCl₂ (20 mM Tris pH 8.1, 40 mM MgCl₂) or 3-μL 20× Mg-ATP (20 mM Tris pH 8.1, 40 mM MgCl₂, 10 mM ATP). A Fluorolog-3 spectrofluorometer (Jobin Yvon) was used for fluorescence measurements. The solution was excited at 435 nm (CFP excitation) and scanned for emission from 450 to 560 nm. FRET/CFP ratios were determined by dividing peak YFP emission (523–525 nm) by peak CFP emission (473–475 nm). Ratios were normalized by setting the average ratio in the absence of ATP and GST-WASP-CA to 1 and then determining the ratios +ATP and +GST-WASP-CA relative to that baseline.