Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Significance

Assembly of actin filaments is tightly regulated to orchestrate basic cellular processes such as motility, division, and differentiation. To control when and where actin filaments assemble, cells rely on actin filament nucleators including the Arp2/3 (Actin-related proteins 2/3) complex, a seven-subunit protein assembly that nucleates branched actin filaments to create dendritic actin networks. Activity of the Arp2/3 complex is controlled by WASP (Wiskott–Aldrich syndrome protein) proteins, which bind directly to it to activate nucleation. To understand how WASP proteins activate the complex, we used chemical cross-linking to engineer an Arp2/3 complex that is locked into an “on” state without WASP. In addition, we used biochemical experiments to determine how WASP proteins stimulate the on state. These results have important implications for understanding how cells control the actin cytoskeleton.

Abstract

The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott–Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a “short-pitch” conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP’s CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP–Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3′s barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.

Proper regulation of the actin cytoskeleton is critical for cells to orchestrate processes such as division, differentiation, motility, and endocytosis. Assembly of new actin filaments is limited by a slow nucleation step, and cells rely on three classes of actin filament nucleators—tandem WH2 domain proteins, formins, and the Arp2/3 (Actin-related proteins 2/3) complex—to control precisely when and where actin filament networks assemble (1, 2). The Arp2/3 complex is unique among actin filament nucleators in that it typically nucleates branched (as opposed to linear) actin filaments (3–5). Branched actin filaments are important for assembling the dendritic networks required for lamellipodial protrusion and endocytosis (6, 7).

The Arp2/3 complex is tightly regulated, and multiple factors, including preformed actin filaments, actin monomers, ATP, and a class of proteins called “nucleation-promoting factors” (NPFs) are typically required for activation (5–7). In some species, phosphorylation of the Arp2/3 complex is also required for activation (8). WASP (Wiskott–Aldrich syndrome protein) family proteins are the best understood NPFs and share a conserved polypeptide sequence called “VCA” (verprolin homology, central, acidic), which constitutes the minimal region sufficient for Arp2/3 complex activation. WASP proteins interact with actin monomers through their V and C regions and bind Arp2/3 complex through the C and A regions (9, 10). How WASP proteins activate the Arp2/3 complex is unknown, but available data suggest they play multiple roles in the branching nucleation reaction. For instance, several lines of evidence indicate that the CA segment of WASP proteins stimulates a conformational change in the complex important for activation (11–15). In addition, WASP-VCA recruits actin monomers to the complex through its V region. Kinetic simulations of the weak NPF-independent activity of the Arp2/3 complex suggested actin monomers must bind the complex to form a nucleus, leading to the idea that WASP-recruited monomers could “jump start” nucleation (16, 17). The observation that the V region is required for WASP-mediated activation of the complex supported this idea and indicated that direct monomer tethering not only might accelerate nucleation but also might be a general requirement for the assembly of the nucleus (9, 10, 12). In addition to its activating influence, WASP-VCA also can have an inhibitory influence on the kinetics of branching nucleation. Specifically, recent single-molecule total internal reflection fluorescence (TIRF) experiments showed that VCA must be released from nascent branch junctions before nucleation and that slow WASP-VCA release limits nucleation rates (18, 19). Efforts to understand the relative contributions of each of these roles of WASP on activation have been hampered by a lack of methods that can disentangle these functions or directly probe the connection between conformation and activity.

Two of the seven protein subunits in the Arp2/3 complex, Arp2 and Arp3, are homologous to actin. High-resolution crystal structures of inactive Arp2/3 complex show Arp2 and Arp3 interacting end-to-end in a “splayed” conformation (20–23). Upon activation, Arp2 is hypothesized to undergo a rigid body movement that moves it ∼25 Å into position next to Arp3, forming an Arp2–Arp3 “short-pitch” dimer that structurally mimics two consecutive actin subunits within a filament (20, 24). Low-resolution EM structures of Arp2/3 complex bound to the WASP proteins N-WASP (neural Wiskott–Aldrich syndrome protein) or WAVE1 (WASP-family verprolin-homologous protein-1) support this hypothesis, because they show Arp2 and Arp3 in a filament-like arrangement (15). In addition, recent experiments demonstrated that N-WASP stimulates site-specific cross-linking of engineered cysteines on Arp2 and Arp3 that react only when the complex adopts the short-pitch conformation (11). These studies suggest a general function of WASP proteins may be to stimulate the short-pitch conformation, providing an explanation for how they could activate the complex. However, whether this function is conserved among WASP proteins and whether adoption of the short-pitch conformation is sufficient for activation is unknown. Importantly, because WASP must recruit actin monomers to the complex for potent activation (9, 12), it is possible that the short-pitch Arp2–Arp3 dimer provides only the initial core of a nucleus to which one or more WASP-tethered actin monomers must be added for nucleation (7). Therefore, whether stimulation of the short-pitch conformation without direct monomer tethering could activate the complex is unknown.

Understanding WASP-mediated activation requires knowledge of both the role of the short-pitch conformation and the mechanism by which WASP proteins could stimulate this conformational change. Despite numerous studies, how the WASP-CA region binds the Arp2/3 complex is still unclear. Biophysical and biochemical experiments, including analytical ultracentrifugation, isothermal titration calorimetry (ITC), and cross-linking indicate that the Arp2/3 complex binds two WASP-CA molecules (25–27). Cross-linking and label-transfer experiments suggest one CA binding site is on Arp3 and that the second spans Arp2 and ARPC1 (Actin-related protein 2/3 complex, subunit 1) (10, 25, 28–30). NMR line-broadening experiments and mutational analyses suggest that the C region forms an amphipathic helix when it binds to the complex (31). Together with homology modeling and recent low-resolution crystal structures, these data suggest that the C helix binds the barbed-end groove of each Arp just as the V region helix binds the barbed-end groove of actin (Fig. 1A) (25–27, 32). Less is known about interactions with A, but a short segment of electron density between subdomains 3 and 4 in Arp3 in a cocrystal structure was interpreted as the conserved tryptophan from the A region of one bound WASP (27). The second A region is thought to bind the ARPC1 subunit, because this subunit in isolation can bind to VCA in a mode dependent on the conserved tryptophan (33). These data, together with distance measurements reported in a recent FRET study (26), have led to an approximate model for CA binding at each site (Fig. 1A). This model is consistent with the majority, but not all, of the published data (15) and provides an important starting point for understanding the molecular basis for WASP-mediated conformational changes. Surprisingly, few studies have used mutational dissection of the complex to investigate WASP binding, and little high-resolution structural informational is available (27, 34), so the model remains tentative. Furthermore, the lack of biochemical dissection leaves open the important question of how the engagement of WASP could stimulate the conformational rearrangement of the complex thought to be required for activation.

Fig. 1.

Hypothesized CA binding mode and schematic of the short-pitch cross-linking assay. (A) Hypothetical model showing proposed binding sites for the WASP family CA region on the Arp2/3 complex. The Arp2/3 complex surface representation is based on the structure of the GMFγ (Glia maturation factor gamma)-bound Arp2/3 complex (PDB ID code 4JD2) (23). (B) Ribbon diagrams of Arp2 and Arp3 in the splayed (Left) and short-pitch (Right) conformations showing the design of the engineered cross-linking assay. The structure of the GMFγ-bound Arp2/3 complex (PDB ID code 4JD2) was used to make both panels. In the right panel, the actin filament structure of Oda et al. (50) was used to move Arp2 into the short-pitch conformation. The engineered cysteines Arp3(L155C) and Arp2(R198C) are highlighted in cyan. The black arrow shows the direction of the ∼25-Å movement required to position Arp2 in the short-pitch conformation. The structure of the chemical cross-linker (BMOE) used in the short-pitch cross-linking assay is indicated. The cross-linking distance between engineered cysteines is 32.5 Å in the splayed conformation and ranges from ∼8–13 Å in different models of the short-pitch conformation. (Also see SI Appendix, Fig. S1).

Here we use a newly developed engineered cysteine cross-linking assay to investigate the role of the short-pitch conformation in Arp2/3 complex activation (11). Cross-linking the engineered cysteines to hold Arp3 and Arp2 in or near the short-pitch conformation bypasses the need for WASP in activation. Remarkably, the complex shows NPF-independent hyperactivity when cross-linked in this conformation. This result indicates that the switch to the short-pitch conformation is a critical activation step and that direct actin monomer recruitment by WASP-VCA is not required for potent activity. Our cross-linking experiments indicate that stimulation of the short-pitch conformation is a conserved feature of WASP proteins and that the CA region is sufficient for this function. However, our data also suggest that the activating effect of the WASP-mediated conformational change may be masked by the slow release of WASP from nascent branch junctions. Using structure-based mutational analysis and WASP-Arp fusion chimeras, we determined one mechanism by which WASP proteins can stimulate movement of the Arp2 and Arp3 subunits into the short-pitch conformation. We show that WASP competes for binding to the barbed-end groove of Arp3 with the Arp3 C-terminal tail, a structural element that forms an allosteric switch responsible for autoinhibition of the complex. These data support a model in which WASP binds the Arp3 barbed-end groove, displacing the Arp3 C-terminal tail to relieve autoinhibition and stimulate the short-pitch conformation.

Results

Stimulation of the Short-Pitch Conformation Is a Conserved Feature of WASP Family NPFs.

Several studies show that WASP family proteins cause conformational changes in the Arp2/3 complex and that these changes are likely important for activation. For example, binding of WASP-VCA to an Arp2/3 complex tagged with fluorescent proteins on ARPC1 and ARPC3 caused a conformational change detected by increased FRET (12). Mutations that blocked the conformational change decreased the activity of the complex (12). Similarly, EM reconstructions of negatively stained Arp2/3 complexes showed the WASP protein Las17 caused structural rearrangements interpreted as repositioning of Arp2 (13, 14). Electron densities from higher-resolution EM reconstructions (∼2.0 nm) of N-WASP– and WAVE1-bound complexes showed a short-pitch arrangement of Arp2 and Arp3 that could template nucleation (15). We reasoned that if stimulation of the short-pitch conformation is important for activation, it should be a conserved function of WASP family proteins. To test this hypothesis, we took advantage of a recently developed cross-linking assay in which engineered cysteines on budding yeast Arp2 and Arp3 cross-link only when the complex is in or very near the short-pitch conformation (Fig. 1B) (11). Treatment of the dual-cysteine engineered Arp2/3 complex with the 8-Å cross-linker BMOE (bis-maleimidoethane) produces a high-molecular-weight band that is cross-reactive with both Arp2 and Arp3 antibodies (Fig. 2A). This band does not form in either of the single-cysteine complexes (Fig. 2A). Furthermore, the engineered cysteines do not significantly influence the activity of the complex, demonstrating that the cross-linking assay can be used to probe for the conformational switch (11). We tested the influence of canonical WASP family members from mammals and budding yeast on cross-linking of the dual-cysteine engineered complex. Each NPF was added at multiple concentrations to reach saturation and remove the influence of binding-site occupancy on the conformation. VCA segments from N-WASP, WAVE1, WASP, and Las17 each potently increased cross-linking (Fig. 2 B and C and SI Appendix, Table S1). In 1-min cross-linking reactions, nearly saturating concentrations of each WASP family protein produced ∼23–32% cross-linked complex, a 7- to 10-fold increase over reactions without an NPF. Multiple lines of evidence indicate that the influence of these NPFs results not from a change in the chemical reactivity of individual engineered cysteine residues but instead from increased population of the short-pitch conformation. First, although WASP binding could influence the pK_a of the cysteine thiol groups and formation of the thiolate, the high concentration of negative charges of the WASP A regions would be more likely to decrease than increase cysteine reactivity (35). Second, a chemically conservative point mutation in N-WASP potently reduces stimulation of the short-pitch cross-linking even at saturation (see below). This residue was previously determined by FRET to influence WASP-mediated conformational changes in the complex (12).

Fig. 2.

Stimulation of the short-pitch conformation is a conserved feature of WASP family NPFs. (A) Single- or two-color Western blots of short-pitch cross-linking reactions containing 1 μM wild-type (wt), dual-cysteine (Cys-Arp), or single-cysteine (Arp2-Cys or Arp3-Cys) Saccharomyces cerevisiae (Sc) Arp2/3 complexes and 25 μM BMOE reacted for 0 or 5 min. (B) Single-color Western blot of 1-min cross-linking reactions containing 1 μM dual cysteine (Cys-Arp), 25 μM BMOE, and 30 μM WASP family protein, as indicated. (C) Quantification of reactions similar to those described in B, except that multiple concentrations were tested. Concentrations of WASP-CA used (expressed in micromolar) are shown in parentheses. Error bars show SE from three reactions.

To understand the structure of the BMOE cross-linked complex better, we used morphing software to move Arp2 from its position in an inactive (splayed) crystal structure [Protein Data Bank (PDB) ID code 4JD2] to its short-pitch position in the low-resolution EM structure of the Arp2/3 complex at a branch junction (24). At each point in the trajectory we calculated the solvent-accessible surface cross-linking distance between the engineered cysteines (36). Given the cross-linker link of ∼8 Å for BMOE, this analysis suggests that the cross-linked complex is either in or very near the short-pitch conformation (SI Appendix, Fig. S1). Therefore, for simplicity, we will refer to the covalently trapped conformation as the short-pitch conformation in the sections that follow. We discuss the possible limitations of this interpretation in Discussion. Given these considerations, we conclude from the data above that diverse WASP proteins stimulate the movement of Arp2 and Arp3 into the short-pitch “filament-like” arrangement. Importantly, N-WASP-CA alone stimulated this conformation as potently as N-WASP-VCA, as is consistent with binding assays showing that CA but not V interacts with Arp2/3 complex (Fig. 2 B and C) (10).

CA Binding to Both Arp2 and Arp3 Is Important for Stimulation of the Short-Pitch Conformation.

Our data indicate that diverse WASP proteins stimulate the short-pitch conformation, suggesting that this structural state is important for activation. To understand better the role of the short-pitch conformation and how it is promoted, we attempted to create a “locked” short-pitch complex by fusing WASP-CA segments onto the C terminus of either Arp2 or Arp3 in the budding yeast Arp2/3 complex. We designed these fusions based on the binding mode previously proposed for WASP-CA on each Arp (Fig. 1A) (25–27). Fusions were made in the context of the engineered cysteine mutations to allow us to test for formation of the short-pitch conformation. We reasoned that this design potentially could create a constitutively activated complex and provides a method to dissect the importance of CA engagement at each binding site. Furthermore, the fused WASP segments lack V regions, conferring two additional advantages. First, the fused CA cannot recruit monomers to the complex, potentially allowing us to isolate the role of CA-stimulated conformational changes in activation. Second, the V region is proposed to block the elongation of nascent branch junctions, necessitating WASP release (32, 37). By not including the V region, we aimed to eliminate the requirement for WASP release in the nucleation reaction. We show below that the fused CA segments engage the complex properly. Unexpectedly, we found that although CA fusion to either Arp2 or Arp3 stimulated the short-pitch conformation, the effect was modest (Fig. 3 A–C and SI Appendix, Table S2). Both CA fusions failed to cross-link to the levels observed for the wild-type complex saturated with free N-WASP-CA. These observations suggest that the binding of WASP-CA to both Arp2 and Arp3 is required to stimulate the conformational switch maximally. To challenge this hypothesis, we added free CA to each of the fusion complexes. Free WASP-CA added to the Arp3-CA fusion complex increased cross-linking to a level nearly identical to that observed with wild-type complex with saturating CA (23 ± 2% versus 26 ± 2%), demonstrating that binding to both sites is required for potent short-pitch stimulation (Fig. 3 A–C). This result also shows that CA fused to Arp3 is nearly fully functional in its ability to stimulate the short-pitch conformation, because the addition of free CA (to saturate the unoccupied Arp2 site) increases cross-linking efficiency to nearly the same level as CA-saturated wild-type complex. The addition of CA to the Arp2-CA fusion complex yielded less short-pitch cross-linking than in CA-saturated wild-type complex (19 ± 2% versus 26 ± 2%), suggesting that the Arp2-CA fusion is only partially functional in stimulating the short-pitch conformation, potentially because of a minor defect in the way the fused CA engages Arp2. We also attempted to create a dual CA-fusion complex, but sporulation of diploid yeast strains containing both Arp3-CA and Arp2-CA chimeras never yielded a haploid strain containing both chimeras in the absence of the wild-type Arps, indicating that the dual CA fusion complex does not support viability.

Fig. 3.

CA binding to both Arp2 and Arp3 is important for stimulating the short-pitch conformation change. (A) Anti-Arp3 Western blots of short-pitch cross-linking assays containing 1 μM wild-type or CA-fusion ScArp2/3 complexes (with dual-cysteine mutations) and 25 μM BMOE reacted for 1 min in the presence or absence of 20 μM N-WASP-CA. (B) Quantification of reactions run under the conditions described in A. Error bars show SE of three reactions. (C) Cartoon summarizing the short-pitch cross-linking results for wild-type and CA fusion complexes with or without N-WASP-CA. The average percentage of short-pitch cross-linked product formed under each condition is indicated. (D) Maximum polymerization rate vs. concentration of wild-type or Arp-CA fusion complexes in 3 µM 15% pyrene actin polymerization assays. (E) Time courses of 3 µM 15% pyrene actin polymerization assays containing 100 nM wild-type or Arp2-CA fusion complex with or without 20 µM N-WASP-CA. AU, arbitrary units.

CA Release from Arp3 Is Likely Important for Nucleation.

We next asked how fusion of CA to the complex influenced its nucleation activity. Specifically, we wondered if the modest increased population of the short-pitch conformation in each CA fusion would result in increased nucleation activity of these complexes. As expected, the Arp2-CA fusion complex showed increased NPF-independent activity in pyrene actin polymerization assays compared with the wild-type complex (Fig. 3D). In contrast, CA fused to Arp3 potently inhibited the complex, even though it stimulated the short-pitch conformation (Fig. 3D). Previous experiments showed that WASP must be released from the nascent branch junction before nucleation (19), so we hypothesized that the decreased activity of the Arp3-CA fusion complex results from slow (or no) CA release. To test this hypothesis, we measured the influence of free CA on the activity of wild-type and Arp2-CA fusion complexes. The addition of free CA blocked the activity of both these complexes in pyrene actin polymerization assays (Fig. 3E). Therefore, these data are consistent with a model in which slow CA release stalls branching nucleation. This result was unanticipated, because previous structural modeling predicted that V (and not CA) sterically blocks actin monomers from associating with nascent branches to initiate elongation, necessitating WASP release (32, 37). Our data suggest that CA alone can block elongation. However, it is important to note that we currently cannot eliminate the possibility that the fusion of CA to Arp3 may inhibit the complex for reasons unrelated to CA release. For instance, one study reported that CA binding to one of the two sites could inhibit the complex from binding to preformed filaments, so fused CA could block activation by inhibiting filament binding (27).

Locking the Arp2/3 Complex into the Short-Pitch Conformation Causes Potent NPF-Independent Activity.

We created the CA fusion complexes to isolate the influence of the short-pitch conformation on the branching nucleation reaction. However, the CA fusions had complicated effects on the reaction, both stimulating the short-pitch conformation and presumably decreasing nucleation by preventing CA release. Therefore, we sought a different strategy to probe the role of the short-pitch conformation. Our cross-linking experiments showed that, even without WASP, a small fraction of the complex adopts the short-pitch conformation (Fig. 2 A and C), consistent with the weak NPF-independent activity of the budding yeast complex (13, 38). Therefore, we reasoned that cross-linking could be used to trap a fraction of the complex in the short-pitch conformation to test its influence on nucleation activity directly. We cross-linked the dual-cysteine complex with BMOE, quenched the reaction at various time points with DTT, and tested the product in pyrene actin polymerization assays without NPFs (Fig. 4). Remarkably, cross-linking potently activated the NPF-independent activity of the complex, demonstrating that adoption of the short-pitch conformation is a critical activation step (Fig. 4). These results were unexpected because, as noted above, direct recruitment of actin monomers by WASP is required for WASP-mediated activation of the Arp2/3 complex (9). Our data demonstrate that, even in the absence of VCA-mediated monomer tethering, the complex is activated once it is locked in the short-pitch conformation.

Fig. 4.

Locking the Arp2/3 complex into the short-pitch conformation bypasses the requirement for NPFs in activation. (A) Western blot of SDS/PAGE gel of cross-linking reactions containing 1 μM dual-cysteine ScArp2/3 complex (Cys-Arp) and 25 μM BMOE. Reactions were quenched with 1 mM DTT at the times indicated. (B) Quantification of data in A. Error bars show SEs from three separate reactions. (C) Time course of pyrene actin polymerization for assays containing 3 μM 15% pyrene-labeled actin and 10 nM dual-cysteine complex cross-linked for the times indicated. (D) Fold activation of the dual-cysteine complex calculated by dividing the maximum polymerization rate at a given cross-linking time by the maximum polymerization rate of the non–cross-linked complex. Data were taken from C. (E) The number of barbed ends created plotted versus time for reactions in C.

The Complex Locked into the Short-Pitch Conformation Is Hyperactivated.

Our data suggest the dominant activating influence of WASP on the kinetics of branching nucleation stems from its influence on the conformation of the Arp2/3 complex. However, WASP-mediated activation of the Arp2/3 complex involves additional steps that also could influence branching rates, including direct monomer recruitment and WASP release from the branch junctions. To understand better the relative contribution of each of these steps to the reaction kinetics, we directly compared the NPF-independent activity of the cross-linked complex and the WASP-activated non–cross-linked complex. To do so, we first separated the cross-linked complex from the non–cross-linked complex using an affinity column charged with GST-tagged Dip1, a recently described Arp2/3 complex activator which binds preferentially to the cross-linked complex (SI Appendix, Fig. S2) (5). This protocol yielded 98% pure cross-linked complex (Fig. 5A). In pyrene actin polymerization assays, 10 nM purified cross-linked complex produced a maximum polymerization rate of 40.6 ± 2.2 nM/s with a time to half-maximal polymerization (t_1/2) of 33.3 ± 5.8 s (Fig. 5 B–D). By comparison, activation of the non–cross-linked complex by the optimal concentration of N-WASP-VCA produced a slower maximum polymerization rate (28.5 ± 1.0 nM/s) and an increased t_1/2 (93.3 ± 5.8 s) (Fig. 5 B–D). These observations led to the surprising conclusion that the cross-linked complex is more active than the N-WASP–activated non–cross-linked complex. In some contexts, dimerizing WASP increases NPF activity (25, 37, 39), so we also compared optimal activation of the non–cross-linked complex by GST-Las17-VCA and leucine zipper (LZ)-dimerized N-WASP-VCA to the activity of the cross-linked complex. Neither of these NPFs activated the non–cross-linked complex as potently as cross-linking (Fig. 5 B and C). The maximum polymerization rate in reactions containing Arp2/3 complex with either GST-Las17-VCA or LZ-N-WASP-VCA was approximately fourfold less than in reactions with the same concentration of short-pitch cross-linked complex. These data demonstrate that the cross-linked complex is hyperactivated compared with the WASP-activated Arp2/3 complex. One explanation for this phenomenon is that the slow WASP-release step is bypassed in the WASP-independent activity of the short-pitch cross-linked Arp2/3 complex. To challenge this model, we tested the influence of free N-WASP-CA on the activity of the cross-linked complex. As predicted, CA inhibited nucleation, supporting a model in which locking the complex into the short-pitch conformation hyperactivates it by allowing it to bypass the slow WASP release step (Fig. 5 E and F). We note that other attributes of the cross-linked complex also may contribute to its hyperactivity. For instance, the WASP-bound Arp2/3 complex may not fully populate the short-pitch state, leading to decreased nucleation rates compared with the cross-linked complex. In addition, if the cross-linked complex requires preformed filaments for nucleation, CA could inhibit it by decreasing actin filament binding (27). However, CA did not block the cross-linked complex from binding filaments in copelleting assays, arguing against this mechanism (SI Appendix, Fig. S3). Finally, inhibition of spontaneous nucleation by WASP also may decrease the maximal polymerization rate even at optimal WASP concentrations, leading to greater activity of the short-pitch cross-linked complex (16).

Fig. 5.

The short-pitch cross-linked complex is more active than the WASP-activated non–cross-linked complex. (A, Left) Coomassie-stained SDS/PAGE gel showing the pre–cross-linked dual-cysteine complex, the post–cross-linking reaction, and the purified cross-linked complex after passing through a MonoQ column and a GST-Dip1 affinity column. (Right) Anti-Arp3 Western blot of purified cross-linked and non–cross-linked samples. (B) Time courses of 3 μM 15% pyrene actin polymerization for reactions containing 10 nM dual cross-linked cysteine ScArp2/3 complex (“xlinked Arp2/3”) or 10 nM non–cross-linked complex with either no NPF (“dual Cys Arp2/3”), 250 nM N-WASP-VCA, or 50 nM GST-Las17-VCA, as indicated. Multiple reaction time courses are shown for the fastest reactions. (C) Maximum polymerization rate versus NPF concentration for reactions containing 10 nM non–cross-linked complex with N-WASP-VCA, GST-Las17-VCA, or LZ-dimerized N-WASP-VCA in conditions identical to those in B. The average maximum polymerization rate for purified cross-linked complex without NPFs is shown as a red circle. Error bars indicate the SD (n = 3). (D) Comparison of maximum polymerization and time to t_1/2 polymerization for 10 nM cross-linked complex versus 10 nM non–cross-linked complex with 250 nM N-WASP-VCA. Error bars indicate the SD (n = 3). (E) Time course of polymerization of a reaction containing 3 μM 15% pyrene-labeled actin, 2.5 nM purified cross-linked complex, and 0–40 μM N-WASP-CA, as indicated. (F) Plot of the maximum polymerization rate versus N-WASP-CA concentration for reactions in E.

Residues in the C Region Influence the Ability of WASP to Stimulate the Short-Pitch Conformational Change.

How CA binding stimulates the movement of Arp2 into the short-pitch position is unknown. As a first step in dissecting the mechanism, we used the cross-linking assay to test the role of specific WASP-CA residues in stimulating the conformational change. The C consensus sequence is characterized by several conserved hydrophobic residues (Fig. 6A) (31). NMR line-broadening experiments and mutational analyses suggest these hydrophobic residues occupy one face of an amphipathic helix that forms when WASP binds the Arp2/3 complex (31). Immediately C-terminal to the proposed amphipathic helix is a conserved arginine, R477 in WASP. The R477K (R478K in N-WASP) mutation is found in patients with Wiskott–Aldrich syndrome and reduces WASP-mediated activation of the Arp2/3 complex (Fig. 6B) (9, 40). This mutation decreased cross-linking by ∼2.5-fold at saturation compared with wild-type N-WASP, demonstrating the importance of this residue in stimulating the short-pitch switch (Fig. 6C and SI Appendix, Fig. S4 and Table S3). Previous data showed that another mutation within the C region, WASP L470A (L471A in N-WASP), decreased activity in polymerization assays and blocked a conformational change measured by FRET, suggesting that this residue also may be important for stimulating the short-pitch conformation (12, 31). Consistent with this hypothesis, the L471A mutation decreased short-pitch cross-linking by approximately threefold compared with wild-type N-WASP-VCA (Fig. 6C). These observations support a model in which contacts with the C region are critical for stimulating the short-pitch conformation and identify two key residues for stimulating this switch.

Fig. 6.

Residues in the C region are important for stimulating the short-pitch conformational change. (A) Sequence alignment of WASP family CA regions. Conserved hydrophobic (green boxes) and basic (red boxes) residues are indicated, and acidic residues in the A region are highlighted in cyan. N-WASP point mutations and deletion constructs investigated in this study are indicated. Bt, Bos taurus; Hs Homo sapiens; Sc, S. cerevisiae. (B) Maximum polymerization rate versus NPF concentration for polymerization reactions containing 3 μM 15% pyrene actin, 10 nM dual-cysteine Arp2/3 complex, and the indicated concentration of each NPF. (C) Results of 1-min short-pitch cross-linking reactions containing 1 μM dual-cysteine Arp2/3 complex, 25 μM BMOE, and the indicated concentrations (in micromolar) of mutant or wild-type N-WASP-VCA. Error bars show the SE from three reactions.

The A region of WASP family proteins is characterized by a conserved tryptophan surrounded by several acidic residues (Fig. 6A). The A region confers most of the Arp2/3 complex binding affinity to WASP proteins (9). The acidic residues in A play an important role in branching nucleation kinetics. Specifically, deletion of acidic residues in N-WASP-VCA increases the affinity of N-WASP for the complex and decreases branching nucleation rates (19), whereas addition of acidic residues to WAVE1 increases nucleation rates (30). To test the influence of the acidic residues in stimulating the short-pitch switch, we created a (monomeric) N-WASP construct with five acidic resides deleted from A. This construct stimulated cross-linking to the same extent as the wild-type N-WASP-VCA (Fig. 6C and SI Appendix, Fig. S4 and Table S3). Therefore, decreased activation of the complex by the N-WASP(ΔDEDED) mutant cannot be explained by a failure to induce the short-pitch conformation and instead is likely caused by the slow release of the N-WASP(ΔDEDED) from branch junctions (19).

CA–Fusion Complexes Suggest the N-WASP C Region Binds to the Barbed-End Grooves of Arp2 and Arp3.

Our data demonstrate that CA regions from diverse WASP proteins stimulate the short-pitch conformation and that residues in the C region are critical for stimulating the switch. However, understanding how C stimulates this conformation requires knowledge of how C interacts with the complex. Based on biochemical and biophysical studies described above, WASP-C has been proposed to interact with the barbed-end grooves of Arp2 and Arp3 (25–27, 32, 34). The C termini of Arp2 and Arp3 terminate between subdomains 1 and 3, filling the back portion of the barbed-end grooves in a position that either overlaps or sits immediately adjacent to the predicted binding sites for C (Fig. 7) (20). This arrangement provides a fortuitous opportunity to test the proposed binding mode. As described above, we created chimeric subunits in which the CA region of N-WASP is fused to the C terminus of either Arp2 or Arp3 (Fig. 7 A–C). Based on the proposed binding mode, the tip of the Arp3 C terminus sterically overlaps the WASP C helix, so we truncated Arp3 by seven residues, removing the tip of the C-terminal tail and connecting the base of the tail to the N terminus of the N-WASP CA region with a short linker (GSG). We reasoned that fused CA would engage Arp3 properly only if the front of the barbed-end grove is the correct binding site for C, as predicted by the model. A similar strategy was used for Arp2, except that the Arp2 C terminus is shorter, so the binding model predicted that truncation of only three residues from the C terminus would allow the C helix to fold into its predicted position in the groove (Fig. 7C).

Fig. 7.

WASP-CA fused to the C terminus of Arp2 or Arp3 blocks the binding of soluble WASP-VCA to the CA-fusion subunit. (A) Sequence alignment of the C terminus of Arp2, Arp3, and actin, showing the design of Arp CA-fusion constructs. Residues conserved in Arp2 and Arp3 (cyan boxes), Arp2 only (pink boxes), or Arp3 only (green boxes) are indicated. Residues in Arp3 that mark the tip (blue line) and base (red line) of the Arp3 C-terminal tail are indicated. The majority of residues from the fused CA segments [indicated by “(CA)”] were omitted from the alignment. (B) Hypothetical model of the Arp3-CA fusion structure. The fused CA sequence is shown in magenta, and the linker is in green. The model was constructed from the crystal structure of the Arp2/3 complex with bound ATP (PDB ID code 2P9K) (22), with the WASP-C region modeled into position based on a WASP-V–bound actin structure (PDB ID code 2A3Z) (32). The A region was placed between subdomains 3 and 4 based on the structure (PDB ID code 3RSE) (27). (C) Hypothetical model of the Arp2-CA fusion structure. The model was constructed from the crystal structure of the GMF-bound Arp2/3 complex (PDB ID code 4JD2) (23), with WASP-C modeled into position as described above. (D) Schematic of the B4M cross-linking assay to probe WASP-CA binding at each site on the complex. (E) Anti-Arp3 Western blot of B4M UV cross-linking reactions containing 1.5 μM dual-cysteine (“wt”), Arp2-CA fusion, or Arp3-CA fusion complexes with 200 μM ATP and with no NPF, 4 μM N-WASP-VCA(T464C)-B4M, or 100 μM LZ-dimerized VCA (LZ-VCA), as indicated. All three complexes harbor the dual-cysteine engineered mutations. (F) Reactions from E analyzed by Western blotting with anti-Arp2 antibody. (G) Quantification of B4M cross-linking reactions as described in E and F. Error bars are SEs from three reactions.

To determine if fused CA segments are properly engaged, we asked if they could compete with VCA for binding to the complex. To do so, we first reacted the bifunctional cross-linker benzophenone-4-maleimide (B4M) with N-WASP-VCA at an engineered cysteine residue (T464C) immediately N-terminal to the C region (Fig. 7D). We used UV radiation to cross-link this adduct, N-WASP-VCA-T464C-B4M, to the wild-type complex, producing VCA cross-linked adducts of both Arp2 and Arp3, as expected based on previously published cross-linking studies (Fig. 7 E and F) (28). Unlabeled VCA competes with N-WASP-VCA-T464C-B4M for cross-linking to the wild-type complex at the Arp2 and Arp3 sites, demonstrating the reaction is specific. Importantly, the Arp3-CA fusion completely blocked N-WASP-VCA-T464C-B4M from cross-linking to Arp3 (Fig. 7 E–G). This result suggests that CA properly engages its site on Arp3 in the fusion chimera, providing support for a model in which the C region binds the Arp3 barbed-end groove. Fusion of CA to Arp3 did not influence N-WASP-VCA-T464C-B4M cross-linking to Arp2, indicating that the Arp3-CA fusion specifically blocks binding of CA to the Arp3 site. We repeated the B4M cross-linking experiments to test whether the Arp2-CA fusion engages the Arp2 CA-binding site. Under our reaction conditions, 67% of Arp2 from wild-type complex cross-linked to N-WASP-VCA-T464C-B4M, whereas only 4.3% of the Arp2 was cross-linked in the Arp2-CA chimera (Fig. 7 E–G). This potent reduction in cross-linking demonstrates that CA fused to the C terminus of Arp2 blocks the Arp2 CA-binding site. Together these experiments support the proposed binding modes, in which the C region of WASP binds the barbed-end groove of either Arp2 or Arp3. The proposed binding modes also are consistent with recent low-resolution X-ray crystal structures of Arp2/3 complex crystals grown in the presence of WASP-CA, which show putative electron density for WASP-C bound to the barbed ends of Arp2 and Arp3 (34).

WASP-C Likely Displaces the C Terminus from the Barbed-End Groove of Arp3 to Relieve Autoinhibition and Stimulate the Short-Pitch Conformation.

How could WASP-CA stimulate the short-pitch conformation? We analyzed crystal structures of inactive Arp2/3 complex in the context of the CA-binding model to identify interactions that could destabilize intersubunit contacts important for holding the complex in the splayed conformation. We also analyzed low-resolution EM models of the Arp2/3 complex at a branch junction to identify WASP-CA interactions that could stabilize the short-pitch conformation (24). These analyses, in conjunction with results from a recent study (41), led us to hypothesize that interactions of WASP-C with the Arp3 barbed-end groove are critical for stimulating the short-pitch conformation. We recently found that the Arp3 C terminus is an autoregulatory switch important for holding the complex inactive in the absence of NPFs. The Arp3 C-terminal tail engages the Arp3 barbed-end groove and occupies a critical position at the splayed Arp2–Arp3 interface (Fig. 8 A and B). The base of the Arp3 C-terminal tail directly contacts Arp2 to stabilize the splayed conformation, whereas the most C-terminal residues in the Arp3 tail (the “tip”) do not contact Arp2 directly but instead fill the front portion of the barbed-end groove (20–22). Deletion of the entire Arp3 C-terminal tail in either budding or fission yeast Arp2/3 complexes stimulates the short-pitch conformation and increases NPF-independent activity (41). Furthermore, mutations that disrupt interactions of the tip of the tail with the barbed-end groove also stimulate the short-pitch conformation, supporting a model in which release of the tip from the barbed-end groove allosterically relieves autoinhibition by destabilizing the splayed Arp2–Arp3 interface (41) (Fig. 8A). Importantly, our CA-fusion data, along with recently published low-resolution crystal structures (34), suggest that WASP-C engages the barbed-end groove of Arp3, where it would overlap with the tip of the C-terminal tail (Figs. 7 and 8 A and B). Therefore, we hypothesized that WASP could activate the complex by displacing the Arp3 C-terminal tail from the Arp3 barbed-end groove. To test this hypothesis, we first asked if the C terminus and WASP-CA compete for binding to the complex. We used fluorescence anisotropy to measure the affinity of rhodamine-labeled WASP proteins for the budding yeast and fission yeast Arp3ΔC complexes. Using a single-site binding model (SI Appendix, SI Materials and Methods and ref. 27), we found the affinity of rhodamine-labeled WASP proteins increased four- to sixfold for the Arp3ΔC mutants compared with wild-type complexes (Fig. 8C and SI Appendix, Fig. S5). To eliminate the influence of the rhodamine label, we ran competition assays to measure binding of unlabeled WASP proteins. These experiments showed that the Arp3ΔC complexes bind to WASP proteins 4- to 13-fold more tightly than wild-type complex (Fig. 8C). Although the fluorescence anisotropy assay does not allow us to distinguish between binding to Arp2 or to Arp3, we took advantage of the observation that truncation of the Arp3 C terminus causes a previously unreactive endogenous cysteine in Arp3, C426, to react in the BMOE cross-linking assay (Fig. 8D). Arp3 C426 lines the barbed-end groove (Fig. 8B), and deletion of the C terminus allows this residue to cross-link with Arp2 to form a non–short-pitch cross-link that migrates below the short-pitch cross-linked Arp3–Arp2 adduct (Fig. 8D and SI Appendix, Fig. S6). We reasoned that if CA binds to the barbed-end groove of Arp3, it could directly block the reactivity of C426. Consistent with this prediction, the addition of CA to the Arp3ΔC complex decreased cross-linking between Arp2 and Arp3 C426 (Fig. 8D). To provide additional evidence that WASP-C binds the barbed end of Arp3, we asked if WASP-C could be directly cross-linked to C426 in Arp3. To this end, we designed a VCA construct [N-WASP-VCA(V468C)] with a cysteine residue immediately N-terminal to C (SI Appendix, Fig. S7). N-WASP-VCA(V468C) formed a disulfide cross-link with C426 in the Arp3ΔC complex in the presence of CuSO₄ (Fig. 8E). The cross-linking was abolished by the addition of cysteine-free N-WASP-CA, indicating that the cross-linking occurs between bound N-WASP-VCA(V468C) and Arp3. These data support a model in which WASP-C competes directly with the Arp3 C terminus to displace it from the barbed-end groove, revealing a structural mechanism by which WASP binding can stimulate the short-pitch conformation. However, we note that in the absence of high-resolution structures of Arp3 with bound WASP-CA, we cannot eliminate the possibility that competition is not direct but instead involves an allosteric link between the WASP-CA–binding site and the Arp3 C terminus.

Fig. 8.

WASP competes with the Arp3 C terminus for binding the barbed-end groove of Arp3. (A) Model for the relief of autoinhibition of the Arp2/3 complex by WASP-CA binding. B, base of the Arp3 C-terminal tail; NBC, nucleotide-binding cleft; T, tip of the Arp3 C-terminal tail. Subdomains of Arp3 and Arp3 are numbered 1–4. (B) Hypothetical model of WASP-CA (magenta) bound to Arp3 showing a clash between WASP-C and the tip of the Arp3 C-terminal tail (yellow). Cysteine 426, a residue that becomes reactive when the C terminus is deleted, is shown as green spheres. The Arp3 ribbon diagram is based on PDB ID code 1K8K, and WASP-C is modeled into barbed-end groove of Arp3 based on the WASP-V–bound actin structure (PDB ID code 2A3Z) (32). (C) Tabulated results from fluorescence anisotropy binding assays. (D) Two-color (anti-Arp3/anti-Arp2, Upper) or one-color (anti-Arp3, Lower) Western blots of cross-linking reactions containing 1.0 μM wild-type or Arp3ΔC or Arp3ΔC(C426A) fusion complexes (each with dual-cysteine mutations) with or without 20 μM N-WASP-CA showing short-pitch (sp) or non–short-pitch (non sp) products. (E) Anti-Arp3 Western blot of VCA cross-linking reactions containing 0.5 μM wild-type, Arp3ΔC, or Arp3ΔC(C426A) S. cerevisiae Arp2/3 complex, 2 μM N-WASP-VCA(V468C), 50 μM CuSO₄,10 mM imidazole (pH 7.0), 50 mM KCl, 50 μM EGTA, 1 mM MgCl₂, and 200 µM ATP with or without 50 μM N-WASP-CA. Note that the Arp2/3 complexes in this assay lack the engineered cysteines for short-pitch cross-linking.

Discussion

Stimulating the Short-Pitch Conformation Is a Critical NPF Function.

Previous models for activation of the Arp2/3 complex ascribed two major functions to WASP: (i) stimulation of an activating conformational change in the complex (11–13, 15) and (ii) direct recruitment of actin monomers to assemble a nucleus consisting of Arp2, Arp3, and one or more actin monomers (9, 10, 12). The relative contributions of each of these functions was unknown. Here we show that locking the Arp2/3 complex into the short-pitch conformation potently activates it without NPFs, demonstrating that direct monomer tethering is not required to assemble the nucleus, as previously anticipated. Therefore, whether direct actin monomer tethering is required for potent activation by any NPF likely depends on the identity and mechanism of the NPF and is not a fundamental requirement of the branching nucleation reaction. Consistent with this hypothesis, WISH/DIP/SPIN90 (WDS) family proteins can potently activate the Arp2/3 complex but lack known actin monomer-binding domains (5). Although little is known about this class of NPFs, we previously used the cross-linking assay to show that WDS protein Dip1 stimulates formation of the short-pitch conformation, and our data here suggest this stimulation is sufficient for Dip1-mediated activation of the complex (5). Like the WDS family proteins, type II NPFs such as cortactin and fission yeast myosin I interact with the Arp2/3 complex and actin filaments but not with actin monomers (37, 42–44). Unlike WDS proteins, type II NPFs are generally weak activators on their own (7). Recent experiments indicate that, at least in the case of cortactin, this weak activity is caused by an inability to deliver monomers directly to the Arp2/3 complex (42). Type II NPFs typically interact with actin monomer-binding proteins that allow them to deliver monomers to the complex indirectly and potently increase their ability to activate (7, 45, 46). Therefore, as with WASP, a requirement for actin monomer recruitment may be programmed into the activation mechanism of type II NPFs. An important future direction will be to determine why actin monomer recruitment is required for potent activation by some NPFs but not by others and how this requirement is structurally encoded into the activation mechanism.

Here we propose a model in which WASP-CA stimulates the short-pitch conformation by displacing the Arp3 C-terminal tail from the barbed-end groove, thereby triggering destabilization of the splayed conformation. Our data indicate that this mechanism must work coordinately with other activation triggers. For example, in a recent study, we showed that ATP bound to the Arp3 nucleotide cleft allosterically disrupts interactions between the Arp3 tail and the barbed-end groove, providing another mechanism to destabilize the splayed conformation (41). Structural features other than the Arp3 C-terminal tail also must be involved in triggering activation. For example, here we show that CA engagement with Arp2 is also important for stimulation of the short-pitch conformation. In addition, phosphorylation of Arp2 near its interface with ARPC4/ARPC2 is required for activation of the complex in some species and is thought to destabilize the splayed conformation (8, 47). Understanding how the Arp2/3 complex structurally integrates each of these activating responses remains an important open question in the field.

The Role of WASP, Actin Monomers, and Actin Filaments in Activation.

Because actin monomers, WASP, and actin filaments are required to activate the complex in most contexts (4, 5, 9, 10), it has generally been assumed that cooperation between each of these factors is required to shift the conformation into the short-pitch conformation, thereby stimulating activation. Our data led us to reevaluate the relationship between activity and conformation. Specifically, the observation that WASP-VCA (or CA) alone can stimulate cross-linking between short-pitch engineered cysteines makes it unclear why WASP-recruited monomers and actin filaments also are required for activation. Because the cross-linking assay conformationally traps the complex, one possibility is that WASP-bound Arp2/3 complex may not populate the short-pitch state significantly, but by cross-linking we amplify a functionally insignificant effect of WASP on the concentration of short-pitch complex. Previous experiments indeed demonstrate that WASP binding is not sufficient to populate the short-pitch conformation fully, because WASP-recruited actin monomers potently increase the amount of short-pitch cross-linking over reactions with WASP alone (11). However, comparisons of the relative amount of complex cross-linked under different conditions, although yielding no information about absolute equilibrium populations, indicate the bias toward the short-pitch conformation in the presence of WASP alone should be functionally significant. Without WASP, only ∼3% of the Arp2/3 complex was cross-linked after 1 min of cross-linking, suggesting that the complex adopts the short-pitch conformation only weakly in the absence of NPF. However, this amount is functionally significant, because the ScArp2/3 complex shows NPF-independent activity in pyrene actin polymerization assays. Therefore, the ∼7- to 10-fold increase in cross-linking stimulated by WASP-CA (or VCA) proteins without actin probably reports on functionally significant increases in the short-pitch conformation. Several previously published biochemical and biophysical experiments agree with this interpretation, because they indicate that WASP alone causes a significant shift in the conformational equilibrium of the complex, presumably toward an activated state (12–15).

A second explanation for the insufficiency of WASP-CA in activation (despite its influence on the conformation of the complex) is that WASP-CA binding stimulates an intermediate conformation close to but distinct from the short-pitch arrangement. Our data cannot eliminate this possibility, because BMOE cross-linking may not necessarily constrain the complex strictly to the short-pitch state, as noted above. From an intermediate WASP-CA–stimulated conformation, WASP-recruited actin monomers and filaments could trigger population of the “fully” short-pitch state. However, recent EM reconstructions of negatively stained NPF-bound Arp2/3 complexes argue against intermediate WASP-bound conformations, because the particles segregated into only two different conformational classes (15). One class corresponded to the inactive splayed conformation seen in crystal structures, and the second (NPF-bound) class produced 3D density consistent with a filament-like (short-pitch) arrangement of Arp2 and Arp3. This study is in contrast with slightly lower-resolution reconstructions of NPF-bound Arp2/3 complexes that indicated a third conformational class corresponding to an intermediate state (13). Thus, the precise structures of WASP-bound complexes and their role in activation remain an important open question.

Finally, another possibility is that actin filaments (and potentially WASP-recruited monomers) could stimulate activating steps distinct from the adoption of short-pitch conformation. In addition to the movement of Arp2 and Arp3 into the filament-like arrangement, the EM branch-junction model describes intramolecular rearrangements within the Arp subunits that may be important for activation and could be stimulated by actin filaments or monomers. This hypothesis is attractive, given recent experiments suggesting that WASP must be released from nascent branch junctions for nucleation to occur (19, 37). Conformational changes initiated by filaments but distinct from the short-pitch switch could help accelerate WASP release, potentially explaining the requirement for filaments in activation. We anticipate that in addition to the engineered short-pitch dual-cysteine Arp2/3 complex, additional biophysical tools and high-resolution structures will be required to dissect the conformational pathway to activation, including the molecular basis for WASP release. We note that our data provide two observations that may be important for understanding the release mechanism. First, although previous studies used single-molecule TIRF to show WASP release triggers nucleation, these studies used only dimeric WASP proteins (19). Experiments here indicate slow detachment of monomeric WASP proteins also might limit branching rates. Second, previous structural models suggested that the WASP V region blocks monomers from associating with nascent branch junctions (32, 37), explaining the requirement for WASP release. Here we show that CA alone stalls nucleation, demonstrating that the structural features that program WASP release into the nucleation mechanism are more complicated than previously anticipated.

Role of WASP Engagement at Each Binding Site.

We show that engagement of WASP-CA with Arp3 is critical for stimulating the short-pitch conformation. However, experiments with the CA fusion complexes revealed that engagement of WASP-CA at the Arp2–ARPC1 site is also important for this conformational change. WASP engaged at this site likely uses a mechanism other than C-terminal tail displacement, because, unlike Arp3, the Arp2 C terminus does not make contacts that stabilize the splayed (inactive) conformation (20–23). ITC binding measurements show a 10-fold difference in the binding affinity at each of the two WASP-CA–binding sites (26, 27), and it will be important to determine how the asymmetry in binding affinities influences the reaction kinetics. We note that our studies indicate that Arp3 harbors the high-affinity site, because the fluorescence anisotropy binding assay measures binding to the high-affinity site (27), and affinities measured by this method increased when competition with the Arp3 C-terminal tail was eliminated. This conclusion is in agreement with other studies indicating that the high-affinity site is on Arp3 (48). However, some experiments point to the Arp2-ARPC1 site as the high-affinity site (26, 27, 33). For instance, a recent ITC study directly demonstrated that WASP delivers actin monomers to the Arp2 site first, suggesting that, at least in the presence of monomers, the Arp2 site is the higher-affinity site (49). Although the complexity of this issue has precluded a clear consensus, we speculate that the preferred binding site may depend on whether WASP is bound to actin monomers.

Materials and Methods

Please see SI Appendix for a complete description of the methods. This description includes construction of budding yeast and fission yeast plasmids and strains harboring Arp2/3 complex mutations; protein purification and labeling; pyrene actin polymerization assays; BMOE, B4M, and CuSO₄ cross-linking assays; actin filament copelleting; and fluorescence anisotropy binding assays.