Abstract
Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeFx bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeFx transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of Pi from the ADP-Pi transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.
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Introduction
Arp2/3 complex is composed of seven protein subunits and nucleates the formation of actin filament branches on the sides of preexisting “mother” actin filaments1. Elongation of these branched actin filament networks powers eukaryotic cell and organelle movements2. Like actin, Arp2/3 complex is an ATPase. ATP hydrolysis and release of the γ-phosphate from Arp2 and/or Arp3 regulate the turnover and mechanical stability of branched filament networks3,4,5,6,7.
High resolution crystal structures of Arp2/3 complex have been available since 20018,9,10. Advances in electron microscopy have improved the resolution of structures of branch junctions from 25 Å11 to 9 Å12, 3.9 Å9,13 and 3.5 Å8. In all these studies the branches were aged, so the ATP bound to the actin and Arps was hydrolyzed and the γ-phosphate dissociated from the active sites (except for ATP bound to Arp2 in the S. pombe Arp2/3 complex8). These structures established the overall architecture of the branch junction and the conformational changes in Arp2/3 complex during branch formation.
Pico-newton forces applied with hydrodynamic flow in microfluidic chambers dramatically increased the rate that branches dissociate from mother filaments7. Newly formed branches are 20 times less sensitive to force than mature branches that have time to dissociate phosphate from the ADP-Pi intermediate in the active sites of the Arp2/3 complex. Similarly, the high affinity phosphate analog, BeFx, also stabilizes branch junctions. These observations raised the question of how the presence of the γ-phosphate in the active site strengthens the branch junction, but the available structures do not provide the answer.
We report two high resolution cryo-EM structures of actin filament branch junctions formed by S. pombe Arp2/3 complex. A 2.7 Å resolution structure with ADP bound to the actin subunits in the mother and daughter filaments, ADP bound to Arp3 and ATP bound to Arp2 reveals several functionally important parts of Arp2/3 complex absent from lower resolution structures. This structure serves as a reference standard for the mature branch junction. A second structure at 3.2 Å resolution has the phosphate analog BeFx bound with ADP in the active sites of Arp3 and ATP bound to Arp2. Compared with the ADP reference structure, the branch junction with ADP-BeFx has a 2° rotation of the outer domain of Arp3 places subdomain 2 (SD2) 0.8 Å closer to the mother filament and buries more surface area between Arp3 and actin subunits in both the mother and daughter filaments. These differences between the ADP-BeFx and the ADP-structures are expected to be associated with phosphate release and may contribute to reducing the stability of aged branch junctions7.
Results
Cryo-EM structure of a mature Arp2/3 complex branch junction at 2.7 Å resolution
We used cryo-electron micrographs of ~400,000 branch junctions in 16,000 images to reconstruct Arp2/3 complex associated with mother and daughter actin filaments at 2.7 Å resolution (Fig. 1; Table 1). This structure confirms the overall architecture of the branch junction and also reveals interactions that stabilize the branch junction not seen at lower resolution8,9,10.
The higher resolution allows unambiguous modeling of the sidechains of most residues in 8 actin subunits of the mother filament, 2 actin subunits of the daughter filament, and all 7 subunits of Arp2/3 complex. All 8 actin subunits and Arp3 have well defined densities in their active sites for ADP and a coordinated Mg2+ ion. The “backdoor” for phosphate release is closed by a hydrogen bond between R177 and N111 in all mother filament actin subunits. The map shows clearly that Arp2 has bound nucleotide triphosphate that is consistent with Mg2+-ATP, although incomplete occupancy of the γ-phosphate suggests that partial hydrolysis and phosphate dissociation may have occurred8.
Features of the interactions between Arp2/3 complex and the mother filament
All subunits in the Arp2/3 complex, except for Arp2 and ARPC5, contact the mother filament directly. The following paragraphs focus on herein identified features of these interactions that were not resolved in previous, lower resolution structures.
At 2.7 Å resolution, Arp3 D-loop residues 39-57 are disordered as in previous structures, but we also visualize weak density for the D-loop C-terminal region (residues 58-67) in the branch junction (Fig. 1b). The Arp3 D-loop density indicates that residues 58-67 interact with the C-terminal α-helices of ARPC2 and ARPC4 and subdomain-3 (SD3) of actin subunit M4 of the mother filament. Arp3 subdomain-2 also makes multiple interactions with subdomain 1 (SD1) of actin subunit M3, including salt bridges between Arp3 R95 and actin E364 and Arp3 R225 and actin D363 (Supplementary Fig. 1A). Additionally, Arp3 Y218 is within hydrogen bonding distance of actin Q360 (Supplementary Fig. 1A).
At higher resolution the sidechain densities for ARPC1 residues 318-330 of the insert helix are sharper, enabling accurate placement of rotamers (Fig. 1c). The ARPC1 insert helix nestles between the C-terminal tail of ARPC2 and SD3 at the barbed end of mother filament actin subunit M3. The interactions are largely hydrophobic, including contacts of ARPC1 F317 with actin I345, ARPC1 F324 with actin L349 and other interactions of ARPC1 residues M327 and L320 and M3 residue I341 (Supplementary Fig. 1B). ARPC1 R321 forms an electrostatic interaction with ARPC2 E303 and a hydrogen bond with M3 S348 (Supplementary Fig. 1C). The high-resolution map lacks density for the linker between the ß-propeller and N-terminus of the insert helix, further evidence that it is disordered.
The higher resolution ARPC2 structure clarifies the sidechain conformations at the C-terminus of ARPC2 and their interactions with ARPC1 (Fig. 1d). ARPC2 also interacts with two mother filament actin subunits (M3 and M5) as well as Arp2 and Arp3. Salt bridges form between ARPC2 K194 and actin M5 E125, ARPC2 R205 and M5 E117, and ARPC2 D202 and M5 K118 (Supplementary Fig. 1D). Residues R306 and K307 in the C-terminal tail of ARPC2, interact with SD1 of actin M3 and also bridge between the N-terminus of actin M3 and Arp2 subdomain 4 (residues 241-247) (Supplementary Fig. 1C).
ARPC3 makes major contacts with the inner domain of Arp3 (buried surface 898 Å2) and the D-loop of Arp2 (buried surface 639 Å2) and buries a modest surface area of 176 Å2 with mother filament subunit M1. The backbone oxygen of ARPC3 residue R94 forms a hydrogen bond with actin M1 residue Q354.
ARPC4 makes numerous contacts with the mother filament (Supplementary Fig. 1E). The C-terminal α-helix (residues 128-165) of ARPC4 interacts with SD1 of mother filament subunit M5 (buried surface 764 Å2) through salt bridge and hydrogen bond interactions. ARPC4 R158 forms salt bridges with residues E83 and D80 in SD1 of M5, which are reinforced by a hydrogen bond network among ARPC4 N154 and actin M5 residues E83 and T126. Furthermore, ARPC4 forms two additional salt bridges with SD1 of actin subunit M5: between ARPC4 R55 and M5 E99; and between ARPC4 residue M5 E100.
The extended N-terminus of ARPC5 (residues 2-27) is better resolved than in previous structures, revealing sidechain interactions with subdomains 3 and 4 of Arp2 (Supplementary Fig. 1F). Salt bridges form between ARPC5 R4 and Arp2 E316 and ARPC5 E13 and Arp2 K318. ARPC5 residue V8 makes a hydrophobic contact with Arp2 F322.
The mother filament is bent where it associates with Arp2/3 complex in the ADP branch junction (Fig. 2). Arp2/3 complex binds on the convex side of the bent mother filament. To measure bending, we generalized methods to quantify bending and twisting of DNA in atomic structures14 for application to other molecular assemblies (Fig. 2). This analysis revealed a net end-to-end bending of ~2.1° over 4 subunits (M2-M5; Fig. 2c, h).
The geometry of this bend indicates a significantly strained, but still thermally accessible, conformation of the mother filament. The majority of filament bending (~1°) occurs between subunits M2 and M3, while the remainder is distributed between subunits M3–M6. Corresponding radius of curvature values between adjacent, bent subunit pairs are in the range of ~0.15–0.3 μm (\(R=s/\Delta {{\theta }}_{i}\), where s = 2.75 nm is the segment length and \(\Delta {{\theta }}_{i}=\)~0.005–0.015 in radians is the end-to-end angular deflection between two subunits). This is associated with a total of ~0.9 kBT in elastic strain energy (Es) within the bent subunits M1 to M4. We estimated the energy per bent subunit pair as \({0.5{L}_{{{{{{\rm{B}}}}}}}s/R}_{i}^{2}\,{k}_{{{{{{\rm{B}}}}}}}T=0.5{L}_{{{{{{\rm{B}}}}}}}\Delta {{\theta }}_{i}^{2}/s\,{k}_{{{{{{\rm{B}}}}}}}T\), where LB is the bending persistence length ≈10 µm15,16,17, and estimated bending values of 1.0° for M2-M3, 0.7° for M3-M4, and 0.4° for M4-M5.
Our analysis of the filament geometry also reveals minor twisting of the mother filament compared with a helically symmetric actin filament (Supplementary Fig. 2A). The twisting varies significantly from one subunit to the next, deviating in a range of +/− ~ 0.5° from the symmetric value (166.7°).
To further assess filament twisting, we estimated the excess end-to-end twist, or ‘cumulative net twist difference’18 from the average twist of the straight filaments, over the length of the filament (Fig. 2j). Excess end-to-end twist values are calculated from the twist values in Supplementary Fig. 2A according to the description given in the Supplementary Notes and are plotted using the same line styles. The cumulative net twist difference is highly sensitive to small changes over a span of many subunits, because the measurement error stays constant with increasing filament length18. Most of the twisting is localized near subunits M2-M4 where bending is observed, and the cumulative twist difference is ~ +0.9° over the full range of the bent filament segment (subunits M1-M6; Fig. 2j). The positive sign of the cumulative twist difference indicates that the filament is slightly over-twisted compared to the straight, symmetric case. The small torsional elastic strain energy (Es « kBT, calculated using a filament torsional persistence length LT value of 10 µm15) associated with these bending deformations is consistent with model predictions of twist-bend coupling in actin filaments19.
For comparison, we also computed the twist (Fig. 2k, Supplementary Fig. 2B) associated with bending of two bare actin structures in ADP-Pi and ADP nucleotide states20. As with the mother filament structures analyzed here, there is a small but significant net overtwist associated with filament bending (Fig. 2k). This value is more than an order of magnitude smaller than that reported from the analysis of these same bare filament structures20.
Interactions between Arp2/3 complex and daughter filament
The high-resolution map reveals features of the interaction of the pointed end of daughter filament subunit (D1) with the barbed end of Arp3. At lower resolution8 the D-loop of actin subunit D1 encircles Y200 of Arp3 (corresponding to actin Y169), but the density of the sidechain of D1 M44 was weak. Our map has a well-resolved densities for the sidechains of D1 M44 and M47, which make hydrophobic interactions with Arp3 (Fig. 3c). The sidechain of M44 fits in a hydrophobic pocket above the Y200-loop of Arp3. Additional interactions between the barbed end of Arp3 and pointed end of D1 are a hydrogen bond between the backbones of D1 M44 and Arp3 G199, and a salt bridge between R62 in SD2 of D1 and D321 in SD3 of Arp3.
The D-loop of daughter filament subunit (D2) wraps around V169 (corresponding to actin Y169) in the barbed end groove of Arp2. The side chain of actin subunit D2 M44 occupies a hydrophobic pocket above the V169-loop and the hinge-helix of Arp3. Further interactions between actin D2 and Arp2 include a backbone hydrogen bond interaction between D2 residue with Arp2 S168, two hydrogen-bonds between D2 D-loop residue H40 with H173 in SD1 of Arp2 and the Nε atom of D2 Q49 with the oxygen of S168 in SD1 of Arp2 and two salt bridges between R62 in SD2 of D2 with Arp2 D288 (corresponding Arp3 D321), and between E241 in the SD4 of D2 with R332 in the SD3 of Arp2.
Cryo-EM structure of Arp2/3 complex with ADP-BeFx in the actin filament branch at 3.2 Å resolution
We stabilized the structure of the branch junction, corresponding to an ADP-Pi intermediate state of the Arp2/3 complex, by including 2 mM BeSO4 and 10 mM NaF in the buffer during daughter filament formation7.
We reconstructed the branch junction with ADP-BeFx from ~200,000 particles in 8500 cryo-electron micrographs. The quality of the 3.2 Å resolution map allowed for the unambiguous placement of most residues in 8 actins and Arp2/3 complex in an atomic model with the same number of residues as the ADP-structure, except for residues 40–64 in the D-loop of Arp3, which were poorly resolved. The branch structure with ADP-BeFx includes all high-resolution features described for the branch structure with ADP, including specific interactions between Arp2/3 complex with the mother and daughter filaments that stabilize the branch junction.
Nucleotides bound to actin and Arp2/3 complex
Density for BeFx at the γ-phosphate position next to Mg2+-ADP is seen in 5 mother filament actin subunits and 1 daughter filament actin subunit (Supplementary Fig. 3). The density for BeFx is weak in the remaining 2 actin subunits (D2 and M6), likely due to their location at the volume periphery where the BeFx would be less well resolved. The map of Arp3 has clear density corresponding to BeFx in the ATP γ-phosphate position (Fig. 4a). This density is absent in the ADP-Arp3 map (Fig. 4b). Similar to the ADP branch junction structure, the reconstruction of the branch junction prepared with BeFx has strong density for γ-phosphate in the active site of Arp2 (Fig. 4c), confirming that Arp2 of S. pombe Arp2/3 complex does not hydrolyze its bound ATP rapidly during branch formation8.
Arp3 phosphate release modulates interactions with the mother and daughter filaments
In the branch junction with ADP-BeFx bound to Arp3, Arp3 is slightly more flattened than Arp3 in branches with bound ADP owing to a difference of ~2° in the angle between outer and inner domains (Fig. 4d–f). This outer domain rotation (Fig. 4d–f), which would be associated with transition from the ADP-BeFx state to the ADP state, moves the D-loop of Arp3 0.8 Å away from the mother filament (Fig. 5).
Mother filament bending
Mother filaments in branches with ADP-BeFx are slightly less bent than those with ADP (Fig. 2, Fig. 6a, b and Supplementary Movie 1). The net bending between subunits M2-M5 is ~1.5° in the ADP-BeFx branch compared with ~2.1° in the ADP branch (Fig. 2c, d, h and Fig. 6c, d). The estimated bending values are 0.6°, 0.7°, and 0.2° for subunits M2-M5. Accordingly, the corresponding total strain energy in the bent segment of the mother filament in ADP-BeFx branch complex (0.6 kBT) is slightly less than the ADP branch complex (0.9 kBT).
Mother filament twisting
Like the ADP sample, the mother filament in the ADP-BeFx branches is over-twisted along with bending (Fig. 2j), at subunits M2-M4. Overtwisting is less pronounced (0.6°) in the ADP-BeFx branch compared with 0.9° in the ADP branch. We also observed this trend in bare, curved actin filaments20 (Fig. 2k). Thus, ADP actin filaments are more compliant in both bending and twisting than ADP-BeFx filaments.
Mother filament interactions
The buried surface area between Arp2/3 complex and mother filament subunits is the same for branches with ADP and ADP-BeFx but the contacts of Arp3 are distributed differently on M4 and M3. In the branch junction with bound ADP contacts of Arp3 with the mother filaments bury 187.4 Å2 on actin subunit M4 and 393.5 Å2 on actin subunit M3, a total of 581 Å2. In the branch junction with ADP-BeFx the total surface area buried between Arp3 and the mother filament is the same (589 Å2), but the Arp3—M4 contact buries a total surface area of 202.3 Å2 and Arp3—M3 contact buries 385.6 Å2. This redistribution is due to the flatter conformation of Arp3 with ADP-BeFx, which keeps the Arp3 outer domain (subdomains 1 and 2) closer to the mother actin subunit M4, while the inner domain (subdomains 3 and 4) is slightly further from the M3 subunit (Figs. 4f and Fig. 6).
Daughter filament interactions
Arp3 buries slightly more surface area (1524 Å2) on daughter filament subunit D1 with ADP-BeFx bound to both Arp3 and actin D1 than with bound ADP (1415 Å2). This larger contact area is largely attributable to the flatter conformation of the outer domain of Arp3 with bound ADP-BeFx that buries 1017 Å2 on the outer domain of actin D1, compared with 912.4 Å2 in the ADP-state. The difference in surface area is associated with specific sidechain and backbone interactions at the daughter filament interface (Fig. 3). With ADP-BeFx a hydrogen-bonding network stabilizes the D1—Arp3 interface (Fig. 3a, b) that is lost due to twisting of Arp3 without phosphate. R177 in the Arp3 W-loop hydrogen-bonds with the backbone of M47 in the D-loop of daughter filament subunit D1 (Fig. 3b), which is supported by a neighboring intra-subunit hydrogen-bond interaction between the backbone nitrogen of G48 and the sidechain oxygen of Q49 (Fig. 3b). These interactions promote contact between Q49 in the D1 subunit D-loop and the backbone oxygen of G199 in Arp3 (Fig. 3b). Table. 1
Mechanical coupling between Arp2 and Arp3 mediated by ARPC3
Subunits ARPC2, ARPC4 and ARPC5 are fixed on the mother filament in the ADP-BeFx and ADP structures, but Arp3 twisting after phosphate dissociation propagates changes to Arp2 and ARPC3 (Fig. 6a–d). Arp2, Arp3 and ARPC3 undergo a concerted hinge-like movement (Fig. 6c, d). ARPC3 moves with the Arp3 D-loop as it repositions away from the mother filament (Fig. 6c, d) inducing a 1° clamshell opening of Arp2 that shifts the Arp2 D-loop away from the mother filament (Fig. 6d, Supplementary Movie 1) and pivots the daughter filament by a small but measurable amount (0.2°) with respect to the mother filament (Fig. 6b, d; Supplementary Movie 1).
Discussion
Previous work established that hydrolysis of ATP and dissociation of phosphate from actin filaments and Arp2/3 complex in branch junctions influences their stability under mechanical force7. Our two structures provide clues about the mechanisms. The high-resolution structure of the branch junction with ADP bound to the actin subunits and the Arps completes the inventory of interactions between Arp2/3 complex and the mother and daughter filaments and serves as the reference standard for other biochemical states. The second structure with BeFx substituting for phosphate to mimic the ADP-Pi intermediate reveals subtle differences in the interactions of ADP-BeFx-Arp3 with the mother and daughter filaments that may stabilize the branch.
The observed bending between subunits of the mother filament in contact with Arp2/3 complex in the branch junction (Fig. 2) indicates that local filament bending is linked to interactions with Arp2/3 complex. Mother filament bending is evident in two earlier structures of branch junctions8,9 (Fig. 2), though it was not reported. Due to conservation of energy and detailed balance, if Arp2/3 complex bends filaments then it binds preferentially to bent filaments rather than straight ones. This explains why convex bends are favored for initiation of branches21. Either physical forces or thermal motions can create gentle bends in ADP- or ADP-Pi filaments with surfaces complementary to Arp2/3 complex. Arp2/3 complex is positioned exactly the same with respect to the curved filament path, but the bend is slightly less with ADP-BeFx bound to the filament and Arp3 than with bound ADP. This difference in curvature could reflect either or both the effect of bound nucleotide on the conformation of Arp2/3 complex, or the higher stiffness of the mother filament with bound ADP Pi17. We note, however, that the stabilizing effects of BeFx on filament debranching7 originate from its binding Arp3 rather than the mother filament, since the mechanical stability of branches under force was identical with ADP- and ADP-BeFx- mother filaments7.
The mother filament also twists locally where Arp2/3 complex binds (Fig. 2j, Supplementary Fig. 2). Concomitant twisting with bending is anticipated given the coupling between filament bending and twisting deformations (twist-bend coupling elasticity) predicted from theory and mathematical modeling of actin filaments19. The structures of mother filaments with bound Arp2/3 complex reported here support the existence of actin filament twist-bend coupling, as do the structures of bent filaments20. However, the reported extent and character of filament twisting for a given bending deformation in bare filaments20 differs substantially from what we measured for branches (Supplementary Fig. 2c–f).
To assess if this difference originates from Arp2/3 complex binding or the analysis methods used to calculate the filament twist, we applied our analysis methods to bare, bent filaments20. Reanalysis with our method yielded a modest twist change of ~0.1-0.4°(Fig. 2k, Supplementary Fig. 2b) similar to what we found in mother filaments bound to Arp2/3 complex (~0.4-0.6°; Fig. 2j, Supplementary Fig. 2A), rather than large twist oscillations (+/− 6°) between adjacent subunits20 (Supplementary Fig. 2c). We are unsure of the precise origins of this difference (see Supplementary Information) but note that inter-subunit twisting fluctuations of 6° would be associated with torsional strain energies of ~20 kBT15, nearly half the energy needed to instantly fragment an actin filament22. Nevertheless, the similar twist behavior obtained by our analysis for actin filaments, bent either spontaneously by thermal energy or by Arp2/3 complex binding (Fig. 2j, k), favors a mechanism in which mother filament twisting at branches originates from twist-bend coupling, an intrinsic property of the actin filament19.
Our 2.7 Å resolution structure of the Arp2/3 branch junction with ADP bound to the actin subunits and Arp3 in Arp2/3 complex reveals several functionally important interactions of Arp2/3 complex with mother and daughter filaments that were not observed at lower resolution. The ARPC1 insert helix interacts with the mother filament through a hydrogen-bond and hydrophobic contacts. The ARPC2 C-terminal helix and tail bridge Arp2 and mother filament subunits through salt bridge and backbone hydrogen bonds. In addition, the ARPC4 C-terminal helix primarily contacts M5 subunit through salt bridges. Collectively, three major interfaces stabilize the binding of Arp2/3 complex to the mother filament through specific salt bridge, hydrogen bond, and hydrophobic interactions.
Branch formation involves activation of free Arp2/3 complex by binding nucleation promoting factors, which favors binding of Arp2/3 complex to the side of a mother filament and the conformational changes that allow Arp2 and Arp3 to nucleate a daughter filament9,13. As the Arps are brought together like two subunits along the short pitch helix of an actin filament, both change from a twisted to a flattened conformation, similar to subunits in actin filaments, and one or both hydrolyze their bound ATP. The Arps are flattened by a rotation of ~24° of their outer domains comprised of SD1 and SD2 relative to the inner domains. Flattening of Arp3 allows the D-loop in SD2 to interact with mother actin subunits M4 and M3 and is associated with a conformation change in the W-loop (residues 169-180) that not only opens a hydrophobic pocket for binding the daughter filament actin subunit D1 (Fig. 6) but also interferes with binding of C-helix of VCA8, as also seen in Arp2/3 complex activated by Dip-19. The dissociation of the VCA C-helix from the Arp3 allows the D1 actin subunit to bind Arp3.
Dissociation of the γ-phosphate of ATP leaves ADP in the active site of both Arps in the branch junction. The rates of hydrolysis and phosphate dissociation are not known but differ depending on the source of Arp2/3 complex. Branches formed by mammalian Arp2/3 complex have ADP bound to both Arps9,13, while branches formed by S. pombe Arp2/3 complex have ADP bound to Arp3 and ATP bound to Arp28. The structures of branches with ATP or ADP-Pi bound to both Arps are not available but are of great interest, since freshly formed branches are more mechanically stable than aged branches7. Therefore, we used BeFx to mimic the ADP-Pi intermediate of S. pombe Arp2/3 complex.
Our structure of the branch junction with BeFx bound to Arp3 and ATP bound to Arp2 revealed that phosphate release from Arp3 is accompanied by a 2° rotation of the Arp3 outer domain, which reduces its flattening. While this structural change is subtle (Fig. 5d–f and Supplementary Movie 1), it supports models in which phosphate release compromises interactions of Arp3 with both mother and daughter filaments that contribute to the lower mechanical stability of mature branches with bound ADP7. We note that there is some ambiguity as to the nucleotide state of Arp2 in our structures. While there is density at the Arp2 gamma phosphate site in both ADP and ADP-BeFx branch structures, this density in the ADP-state structure seems to be weaker than would be expected for ATP, suggesting that ATP in the Arp2 site may be partially hydrolyzed8 or, in the presence of BeFx, partially substituted by this nucleotide analog. Further experiments are required to resolve this question.
The conformational changes observed here may be general and apply to the Arp2/3 complex from other organisms. The nucleotide-dependent structural changes reported here for pombe Arp2/3 complex result from hydrolysis and phosphate release in Arp3 only, but are concerted and long-range (e.g., ATPase of Arp3 the affects conformation of Arp2). Accordingly, hydrolysis and phosphate release by Arp2 could potentially induce additional long-range conformational changes in the complex, possibly explaining why the ATPase of Arp2 and/or Arp3 promotes dissociation of Arp2/3 complex branches in other organisms5,13,23.
Implications for GMF-mediated debranching of actin filaments: The glial maturation factor (GMF) rapidly dissociates S. pombe ADP-Arp2/3 complex branches with half maximal concentration of 40 nM7. One the other hand, GMF concentrations up to 1 μΜ failed to dissociate branches formed by S. pombe Arp2/3 complex with bound ADP-BeFx7, suggesting weak binding of GMF to branches with bound ATP or ADP-Pi. Similarly, GMF has a higher affinity for mammalian ADP-Arp2/3 complex in solution than ATP-Arp2/3 complex24.
Since BeFx binds and affects only Arp3 in the S. pombe complex, it is appealing to consider a mechanism in which GMF triggers debranching by preferentially binding the partially flattened ADP-Arp3 conformation. By analogy with cofilin binding to actin filaments25,26,27, GMF binding is likely limited by site accessibility and conformational fluctuations of Arp3. These fluctuations presumably include ‘unflattening’ but must be larger than the 2° rotation observed here to account for the several order of magnitude change in GMF binding affinity7, suggesting nucleotide-dependent, conformational dynamics of the Arp2/3 complex govern GMF binding. Also, by analogy with cofilin binding to actin filaments28, subsequent GMF occupancy could favor conformational changes at the pointed end of Arp3 that weaken the Arp2/3 complex-mother interface and promote debranching.
This mechanism, while it ascribes significance to the structural changes of Arp3, is at variance with GMF binding Arp2 of unbranched B. taurus Arp2/3 complex in solution29. We note, however, that the structural changes originating in Arp3 observed here are equally consistent with a mechanism in which phosphate release from Arp3 promotes GMF binding to Arp 2 through linked conformational changes propagated from Arp3 to Arp2 through ARPC3. A concerted rigid-body rotation of Arp2 and ARPC3 triggered by Arp3 unflattening (Fig. 6c, d) slightly reorients the Arp2 D-loop, potentially facilitating GMF binding to the Arp2-D2 interface. Molecular modeling of completely un-flattened Arp2 and Arp3 conformations as seen in the GMF-Arp2/3 complex29 suggest that ARPC3 mediated coupling between Arp2 and Arp3 are maintained for larger conformational changes, such as those associated with GMF binding to Arp2 (Fig. 6e and Supplementary Movie 2). Accordingly, GMF binding to Arp2 is expected to unflatten both Arp2 and Arp3, thereby weakening the mother and/or daughter filament interfaces and promoting debranching.
We cannot eliminate the possibility that GMF binds to different Arps in Arp2/3 complex depending on the organismal source and/or in solution vs in branches, nucleotide state(s), or that multiple GMF binding events (e.g., to Arp2 and Arp3) may promote debranching30.
Methods
Protein purification: Actin was purified from rabbit skeletal muscle acetone powder as described7. S. pombe Arp2/3 complex, GCN4-VCA and capping protein were purified as described7,8. Arp2/3 complex was stored in QB buffer (10 mM PIPES pH 6.8, 1000 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM ATP and 1 mM DTT)8.
Assembly of branched junctions: Branched actin filaments were prepared as described8. Briefly, actin monomers with bound Ca2+ were converted to Mg2+-actin by equilibrating with 50 μΜ MgCl2 and 0.2 mM EGTA (pH 7.5) for 10 min on ice. Actin was polymerized in the presence of capping protein (CP) by sequentially mixing 8.75 μΜ Mg-ATP-actin monomers with 0.75 μΜ CP and equilibrated at room temperature for 1 h. In parallel, Arp2/3 complex (0.4 μΜ) in QB buffer was activated by mixing 0.85 μΜ GCN4-VCA and 50 µM ATP and incubated at 4 °C for 1 h. The capped actin filaments sample was then gently mixed with an equal volume of activated Arp2/3 complex sample using cut pipette tips and equilibrated at 4 °C for 5 min. Daughter filaments were formed and elongated in the presence of CP by adding 0.25 μΜ Mg-actin monomers, 50 μΜ ATP, and 40 nM CP and incubated for 5 min at room temperature. This step was subsequently repeated 4 more times, then aged for ~90 min before preparing grids for cryo-EM data collection7. For Arp2/3 complex-ADP-BeFx samples, a final concentration of 2 mM BeFx (prepared using 2 mM BeSO4 and 10 mM NaF)7 was added during the 4th step of daughter filament formation (as mentioned above).
Sample Freezing and data collection: The samples were frozen on Quantifoil 1.2/1.3 300-mesh (Holey carbon) Au grids that were not glow-discharged. A sample of 3.0 μL was applied onto the carbon side of the grid using FEI VitrobotTM Mark IV at 4 °C and 100% humidity. The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (Ø.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were plunged into liquid ethane at ~180 °C with a wait time of 0.5 s. The vitrified grids were screened for sample homogeneity and ice thickness in a Glacios 200 kV transmission electron microscope equipped with Gatan K2 summit camera. Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe. A defocus value between −2.5 μm and −1.2 μm on a K3 Gatan summit camera in super-resolution mode was used to collect 1 movie per hole (using serialEM data collection software). Each movie contains 41 frames with a frame time of 0.08 s. A dose rate of 28.4 counts/pixel/s and a physical pixel size of 1.346 Å was used. In total, we collect ~16,000 movies of the ADP-state and 8519 movies of the ADP-BeFx state. Approximately half of the movies of the ADP-BeFx state were collected with the grids (alpha-tilt) normal to the beam and half with the grid tilted 20°.
EM Image processing: Both ADP and ADP-BeFx datasets were processed entirely using CryoSPARC v331. Micrographs were subjected to motion correction (Patch Motion Correction) and CTF estimation before particle picking. About 3500 particles that contained a “Y-shaped” branch junction were picked manually using a box size of 256 Å. Manually picked particles included a variety of daughter filament orientations, such that their angles with respect to the optical axis ranged from 0 to 90 degrees. Special care was taken to include rare views where the daughter filaments were parallel (nearly parallel) to the optical axis. The manually picked particles were then subjected to 2-dimensional classification to create templates for template-based particle picking. The resulting template-based particle picking method (box size of 256 Å) identified ~3 million particles that contained junction particles, filament particles and other non-protein particles. These particles were subjected to another round of 2-D classification to separate junction particles from other filamentous particles. The resulting junction particle templates were subjected to iterative rounds of particle-picking and 2D-classification (Supplementary Fig. 5) to identify classes containing junction particles. The last iteration was followed by several more rounds of 2D-classification, yielding a total of ~1.7 million particles assigned as junction and filament particles. The final classification included several classes with daughter filaments that were aligned with the optical axis (2D classes, Supplementary Fig. 5) thus ensuring a complete 3D reconstruction without missing angle issues. These particles were then subjected to 3-dimensional structure refinement and classification using “Ab-initio 3D reconstruction” (10 classes) (Supplementary Fig. 5). For both ADP and ADP-BeFx samples, output structures from this 3D refinement/classification included a single junction class containing, ~400,000 junction particles (ADP-state) and ~200,000 particles (ADP-BeFx state). The particles were then subjected to homogeneous refinement, resulting in a 3.2 Å and 3.5 Å resolution map for the two samples respectively. This step was followed by CTF refinement (local and global), that further improved the resolution of the reconstruction to 3.0 Å and 3.3 Å respectively. Next, local motion correction was performed on the particles to further improve the resolution—which resulted in final reconstructions with resolutions of 2.7 Å for the ADP samples and 3.2 Å for the ADP-BeFx samples (Supplementary Fig. 6).
Model building and structure refinement: The map resolution in both states allowed us to build most residues and side chains unambiguously. The recent cryo-EM structure of the Arp2/3 complex in the branch junction at 3.5 Å resolution (PDB: 8E9B)8 was used as the starting model for both structures. The atomic model was subjected to interactive all atom molecular dynamics flexible fitting run using ISOLDE32. Additional modeling was performed using COOT33 and ISOLDE, followed a final refinement using Phenix34. Structures were validated using the “comprehensive validation (cryo-EM)” tool in Phenix34. Structural analysis (RMSD and buried surface area) and visualization was performed using ChimeraX35. Figures were generated using ChimeraX35.
Reference frames for filament bending and twisting estimates: We utilize the methods and notation applied by Britton et al. to DNA14, here adapted for arbitrary filaments such as actin. The reference frame for each filament subunit in a symmetric atomic filament structure (Fig. 2a, left side) is defined as follows: the origin o lies on the helical symmetry axis, the unit vector n follows the helical symmetry axis and s points from o toward the center of mass c of the reference subunit. To track distortions in a helical structure, the atomic coordinates of the reference subunit are aligned by least-squares superposition to individual filament subunits i (red arrows), carrying along the reference coordinate frame to define the subunit coordinate frame oi, si, li, and ni. In a distorted filament, deviations from symmetry cause misalignments between coordinate frames, as shown. This path definition allows the quantification of filament bending and twisting in the presence of other distortions like shearing between subunits14. Here, filament twist is defined as the axial rotation Ti (red)14 undergone between coordinate frames i and i + 1, taking into account any change in path direction that may occur (e.g., if ni is different than ni +1); see Fig. 2 and Supplementary Fig. 2.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
Atomic coordinates and corresponding cryo-EM density maps, including the half maps, masks and FSC curves used to estimate spatial resolution have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Resource (EMD) under the accession codes 8UXW/ EMD- 42787 (ADP Arp2/3 branch complex), 8UXX/ EMD- 42788 (ADP BeFx Arp2/3 branch complex), 8UZ0/ EMD-42829 (straight F-actin from ADP Arp2/3 sample), and 8UZ1/EMD-42830 (straight F-actin from ADP BeFx Arp2/3 sample). Source data are provided with this paper.
Code availability
A python script, generalized for arbitrary polymer atomic structures, was written for UCSF ChimeraX to perform filament twisting and bending analyses and visualizations in Fig. 5 and Supplementary Fig. 5. The script is publicly available on GitLab (https://gitlab.com/cvsindelar/fil_twistbend3d). A second UCSF ChimeraX python script was written to generate domain rotation and shifting analyses, and associated visualizations in Figs. 5, 6 and S4. This script is also available on gitlab (https://gitlab.com/cvsindelar/subdomain_transforms).
Change history
15 March 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41467-024-46804-9
References
Mullins, R. D., Heuser, J. A. & Pollard, T. D. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl Acad. Sci. USA 95, 6181–6186 (1998).
Molinie, N. & Gautreau, A. The Arp2/3 regulatory system and its deregulation in cancer. Physiol. Rev. 98, 215–238 (2018).
Dayel, M. J., Holleran, E. A. & Mullins, R. D. Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments. Proc. Natl Acad. Sci. USA 98, 14871–14876 (2001).
Dayel, M. J. & Mullins, R. D. Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2. PLoS Biol. 2, E91 (2004).
Ingerman, E., Hsiao, J. Y. & Mullins, R. D. Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly. J. Cell Biol. 200, 619–633 (2013).
Le Clainche, C., Pantaloni, D. & Carlier, M. F. ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays. Proc. Natl Acad. Sci. USA 100, 6337–6342 (2003).
Pandit, N. G. et al. Force and phosphate release from Arp2/3 complex promote dissociation of actin filament branches. Proc. Natl Acad. Sci. USA 117, 13519–13528 (2020).
Chou, S. Z., Chatterjee, M. & Pollard, T. D. Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structureof the branch junction. Proc. Natl Acad. Sci. USA 119, e2206722119 (2022).
Shaaban, M., Chowdhury, S. & Nolen, B. J. Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state. Nat. Struct. Mol. Biol. 27, 1009–1016 (2020).
Robinson, R. C. et al. Crystal structure of Arp2/3 complex. Science 294, 1679–1684 (2001).
Rouiller, I. et al. The structural basis of actin filament branching by the Arp2/3 complex. J. Cell Biol. 180, 887–895 (2008).
Fassler, F., Dimchev, G., Hodirnau, V. V., Wan, W. & Schur, F. K. M. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. Nat. Commun. 11, 6437 (2020).
Ding, B. et al. Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy. Proc. Natl Acad. Sci. USA 119, e2202723119 (2022).
Britton, L. A., Olson, W. K. & Tobias, I. Two perspectives on the twist of DNA. J. Chem. Phys. 131, 245101 (2009).
Bibeau, J. P. et al. Twist response of actin filaments. Proc. Natl Acad. Sci. USA 120, e2208536120 (2023).
McCullough, B. R., Blanchoin, L., Martiel, J. L. & De la Cruz, E. M. Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics. J. Mol. Biol. 381, 550–558 (2008).
Isambert, H. et al. Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins. J. Biol. Chem. 270, 11437–11444 (1995).
Huehn, A. et al. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments. J. Biol. Chem. 293, 5377–5383 (2018).
De La Cruz, E. M., Roland, J., McCullough, B. R., Blanchoin, L. & Martiel, J. L. Origin of twist-bend coupling in actin filaments. Biophys. J. 99, 1852–1860 (2010).
Reynolds, M. J., Hachicho, C., Carl, A. G., Gong, R. & Alushin, G. M. Bending forces and nucleotide state jointly regulate F-actin structure. Nature 611, 380–386 (2022).
Risca, V. I. et al. Actin filament curvature biases branching direction. Proc. Natl Acad. Sci. USA 109, 2913–2918 (2012).
Schramm, A. C., Hocky, G. M., Voth, G. A., Martiel, J. L. & De La Cruz, E. M. Plastic deformation and fragmentation of strained actin filaments. Biophys. J. 117, 453–463 (2019).
Martin, A. C., Welch, M. D. & Drubin, D. G. Arp2/3 A. T. P. hydrolysis-catalysed branch dissociation is critical for endocytic force generation. Nat. Cell Biol. 8, 826–833 (2006).
Boczkowska, M., Rebowski, G. & Dominguez, R. Glia maturation factor (GMF) interacts with Arp2/3 complex in a nucleotide state-dependent manner. J. Biol. Chem. 288, 25683–25688 (2013).
Blanchoin, L. & Pollard, T. D. Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments. J. Biol. Chem. 274, 15538–15546 (1999).
Cao, W., Goodarzi, J. P. & De La Cruz, E. M. Energetics and kinetics of cooperative cofilin-actin filament interactions. J. Mol. Biol. 361, 257–267 (2006).
De La Cruz, E. M. & Sept, D. The kinetics of cooperative cofilin binding reveals two states of the cofilin-actin filament. Biophys. J. 98, 1893–1901 (2010).
Huehn, A. R. et al. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments. Proc. Natl Acad. Sci. USA 117, 1478–1484 (2020).
Luan, Q. & Nolen, B. J. Structural basis for regulation of Arp2/3 complex by GMF. Nat. Struct. Mol. Biol. 20, 1062–1068 (2013).
Ydenberg, C. A. et al. GMF severs actin-Arp2/3 complex branch junctions by a cofilin-like mechanism. Curr. Biol. 23, 1037–1045 (2013).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr D. Struct. Biol. 74, 519–530 (2018).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D. Biol. Crystallogr 66, 486–501 (2010).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D. Biol. Crystallogr 66, 213–221 (2010).
Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Acknowledgements
Research reported in this publication was supported by National Institute of General Medical Sciences of the National Institutes of Health under award numbers R35GM136656 to EMDLC, R01GM026338 to TDP, and R01GM110530 to CS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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S.S.C. designed experiments, prepared cryo-EM samples, collected data, performed 3D reconstructions and digital image data analysis, modeled and analyzed atomic structures and cowrote the first draft of the manuscript. S.Z.C. designed experiments, prepared samples, collected data and assisted with sample preparation. W.C. contributed to the twist/bend analysis and edited the manuscript. T.D.P. designed experiments, cowrote and edited the manuscript, and supervised the project. E.D.L.C. designed experiments, cowrote and edited the manuscript and supervised the project. C.V.S. designed experiments, modeled and analyzed atomic structures, modeled and analyzed structures, performed bending and twisting analysis, cowrote and edited the manuscript and supervised the project.
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Chavali, S.S., Chou, S.Z., Cao, W. et al. Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex. Nat Commun 15, 2059 (2024). https://doi.org/10.1038/s41467-024-46179-x
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DOI: https://doi.org/10.1038/s41467-024-46179-x
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