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Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state

Nature Structural & Molecular Biology volume 27, pages 1009–1016 (2020)Cite this article

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Abstract

Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.

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Fig. 1: Overview of structures of inactive Arp2/3 complex and activated Arp2/3 complex bound to the actin filament pointed end and Dip1.
Fig. 2: Arp2 and Arp3 flatten and adopt the short-pitch helical conformation upon activation.
Fig. 3: Arp2 and Arp3 make filament-like long-pitch interactions with actin.
Fig. 4: Nucleotide-binding states of the Arps and actin.
Fig. 5: Dip1 bends the ARPC4 long helix to twist the clamp and rotate a block of subunits into the short-pitch conformation.
Fig. 6: Proposed mechanism of Arp2/3 complex activation by Dip1.

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Data availability

EM maps and atomic models were deposited in the Electron Microscopy Data Bank and wwPDB, respectively, with accession entries EMD-21502 and PDB 6W17 (Dip1–Arp2/3–actin) and EMD-21503 and PDB 6W18 (inactive Arp2/3 complex).

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Acknowledgements

Cryo-EM data were collected at the Stony Brook University (SBU) Cryo-EM center, and we thank G. Hu for providing support at this center. Computational infrastructures were provided by the cryo-EM and High-Performance Computing facilities at SBU. We thank N. Samaroo and B. Ding for help with experiments, and acknowledge K. Prehoda and members of the Nolen and Chowdhury laboratory for comments on the manuscript. We also thank G. C. Lander for providing access to the Scripps Research cryo-EM facility for determining the feasibility of the project. We thank D. Kovar for the capping protein expression vector. This work was supported by SBU start-up funds to S.C., and by NIH grant nos. R01GM092917 and R35GM136319 to B.J.N. The SBU cryo-EM center is supported by NIH grant no. S10 OD012272. M.S. was supported by the Fulbright Association.

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Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA

    Mohammed Shaaban & Saikat Chowdhury

  2. Institute of Molecular Biology and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA

    Brad J. Nolen

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Contributions

B.J.N. and S.C. conceived the project. Biochemical conditions for preparation of samples were determined by B.J.N., S.C. and M.S. Cryo-EM data collection and data processing were performed by M.S. and S.C. Atomic models were built by M.S. B.J.N performed structural analysis. All authors participated in manuscript preparation.

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Correspondence to Saikat Chowdhury or Brad J. Nolen.

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Extended data

Extended Data Fig. 1 Cryo-Electron Microscopy of Arp2/3 complex in active and inactive states.

a, A representative micrograph containing actin filaments polymerized from Dip1-activated Arp2/3 complex. Circles mark the knob of density corresponding to Dip1-bound S. pombe Arp2/3 complex at the pointed ends of filaments. Dip1 and Arp2/3 complex bind weakly to the ends of spontaneously nucleated actin filaments but strongly to Dip1–Arp2/3–nucleated filaments34, so we concluded that most or all filaments with density for Arp2/3 complex were nucleated by Dip1-activated Arp2/3 complex. b, Reference-free 2D class averages obtained from particles corresponding to the knob shaped densities highlighted by circles in a. These class averages show Dip1–Arp2/3–actin-filament complex adopts different orientations in vitrified ice. c, Two different views of the reconstructed 3D map of active Arp2/3 complex bound to Dip1 and pointed end of nucleated actin filament, colored based on local resolution values of each voxel. d, Euler angle distribution plot for particles contributing to the reconstructed map in c (map colored in gray at the center). e, A micrograph of S. pombe Arp2/3 complex sample collected by tilting the stage to 40˚ showing uniform distribution of particles in ice. f, Representative reference free 2D class averages of Arp2/3 complex showing different orientations of the complex obtained from particles extracted from tilted and non-tilted micrographs. g, Two different rotated views of the 3D reconstruction of Arp2/3 complex in an inactive state colored based on per-voxel local resolution values. h, Plot showing the Euler angle distribution of particles that went into the final reconstructed inactive Arp2/3 complex map (colored in gray). Scale bars (white line) for the micrographs in a and e represent distance of 25 nm and for the 2D class averages b and f represent 12.5 nm distance.

Extended Data Fig. 2 Single-particle cryo-EM data processing workflow for 3D reconstructions of activated and inactive Arp2/3 complex.

a, Data processing schematic for Dip1–Arp2/3–actin-filament complex. Particle stack was initially binned by factor of four and subjected to multiple rounds of 2D and 3D classification to discard particles that did not correspond to the complex. The cleaned particle stack was re-extracted without binning and further subjected to downstream 3D processing. All 3D classifications were performed without image alignment (referred to as clustering). Final composite map was generated by combining focused maps corresponding to the Dip1-Arp2/3 cap (Region-1, colored purple) and the Arp-actin filament (Region-2, colored blue). b, Schematic for processing of Arp2/3 complex (inactive state) cryo-EM data. Particles extracted from all micrographs (tilted and non-tilted) were cleaned by multiple rounds of 2D classification and then subjected to multiple ab initio 3D reconstructions. Particles corresponding to intact Arp2/3 complex were further subjected to downstream data processing that lead to a 4.2 Å 3D reconstruction of Arp2/3 complex in inactive state.

Extended Data Fig. 3 Reconstructed maps and atomic models of subunits.

Fourier shell correlation (FSC) plots between masked (yellow) and unmasked (orange) half maps, and final map and model (blue) for global resolution estimates for a, Dip1–Arp2/3–actin-filament complex, and b, inactive Arp2/3 complex. EM density for each subunit is shown as gray mesh. c, Arp3, d, Arp2, e, ARPC1, f, ARPC2, g, ARPC3, h, ARPC4, i, ARPC5, j, Dip1, and k, actin maps with built models. l, Maps and models corresponding to the long helices from the ‘clamp’ subunits, ARPC2 (cyan) and ARPC4 (blue) in the inactive state (left) and active state (right).

Extended Data Fig. 4 The structure of inactive S. pombe Arp2/3 complex is nearly identical to other inactive Arp2/3 complex structures.

a, Structural superposition of inactive S. pombe Arp2/3 complex (colored subunits) from the cryo-EM reconstruction presented here with inactive Bos taurus Arp2/3 complex (semi-transparent gray subunits, PDB 4JD218). Structures were superposed using Arp3 subdomains 1 and 2, Arp2, ARPC1, ARPC2 and ARPC4. b, Structural superposition showing that Arp3 from the S. pombe inactive Arp2/3 complex structure shows a more open nucleotide binding cleft than other ATP-bound Arp3 structures. Subdomains 1 and 2 of inactive S. pombe Arp3 (orange) were superposed on inactive BtArp3 (transparent gray, PDB 4JD2). Cyan arrow shows the direction of rotation of subdomains 3 and 4 toward subdomains 1 and 2 during cleft closure. Red arrow shows the axis of rotation of cleft closure. A rotation of 10.3° about this axis is required to close the S. pombe nucleotide cleft to the same extent as the BtArp2/3 structure. The open cleft in the inactive S. pombe Arp2/3 complex is the major structural difference between the two structures.

Extended Data Fig. 5 Comparison of Arp2 and Arp3 in the activated complex to actin in the nucleated filament.

a, Superposition of Arp3 Cα atoms from the active structure to actin subunit n in the nucleated filament. The RMSD between 256 pruned atom pairs is 1.31 Å (across all 354 pairs: 3.45). b, Superposition of Arp2 Cα atoms from the active structure to actin subunit n in the nucleated filament. The RMSD between 314 pruned atom pairs is 0.92 Å (across all 352 pairs: 2.74).

Extended Data Fig. 6 Partial flattening allows Arp3 to make short pitch interactions that mimic those of a short pitch actin dimer.

a, Left, Arp3 from the active (orange) and inactive (semi-transparent gray) Arp2/3 complex structures, with definition of measurements in plot. Top, the twist angle (𝜑) is the dihedral angle between the centers of mass (blue spheres) of each of the four subdomains (see Methods). Bottom, distance x1 between Y92 Cα and K235 Cα in S. pombe Arp3. Right, plot of the twist (𝜑) versus distance x1 in inactive or active structures of actin, Arp2 or Arp3. Data points highlighted in yellow are from the activated Arp2/3 complex structure. A subset of structures are labeled: em (from cryo-EM reconstructions here); PDB 6DEC (Spin90 bound to Bos taurus (Bt)Arp2/3 complex, chain ID in parentheses21); PDB 4JD2 (BtArp2/3 complex bound to GMF18); PDB 1NWK and PDB 1J6Z (representative actin monomer structures bound to ATP or ADP, respectively53,54). b, Superposition of inactive (twisted) Arp3 from the inactive S. pombe Arp2/3 complex cryo-EM structure and Arp3 from the activated structure overlaid using subdomains 3 and 4. Arp2 in the short pitch conformation from the activated structure is shown in pink. Partial flattening of Arp3 upon activation allows subdomains 1 and 2 to make closer contacts with Arp2 in the short pitch conformation. c, Surface area buried at the short pitch interface between Arp2 and Arp3 in the active structure (model 1) or theoretical models in which the Arps are modeled in the short pitch conformation and either Arp2 (model 2), Arp3 (model 3), or both (model 4) adopt the twisted conformation. Models 2, 3 and 4 were constructed by individually superposing either Arp2, Arp3, or both subunits from the inactive structure onto their corresponding subunit in the activated structure using Cα atoms from subdomains 3 and 4. d, Comparison of the short pitch interface between Arp3 and Arp2 (left panel) or actin (n) and actin (n + 2) right panel. The 9° rotation of Arp2 brings the helix harboring E231 from Arp2 closer to subdomain 1 of Arp3, allowing E231 to interact with R418.

Extended Data Fig. 7 Uncurling of the W-loop facilitates long-pitch interactions with actin.

a, Close up of long pitch interactions between the Arp3 barbed end groove (BEG) and the actin D-loop. Activated Arp3 (orange) is overlaid on an inactive (transparent) structure using subdomains 3 and 4. A structure of Bos taurus Arp2/3 complex (PDB 4JD2) was used as the inactive structure in this and all analyses in this figure as some sidechains at the barbed end of Arp3 are missing in the inactive S. pombe Arp2/3 complex structure presented here. b, Long pitch interactions between the Arp2 BEG in the activated (pink) or inactive (transparent) structure (PDB 4JD2). Arp2 from each structure was superposed using subdomains 3 and 4. c, Structure of Arp3 from activated Arp2/3 complex showing the definitions of the measurements made in panel d. d, Plot of the twist angle (𝜑) versus distance x2—which measures uncurling of the W loop—for inactive or active structures of actin, Arp2 or Arp3. em: structures from the cryo-EM reconstructions presented here; PDB 6DEC: structure of Spin90 bound to Bos taurus (Bt)Arp2/3 complex26; PDB 4JD2: BtArp2/3 complex bound to GMF18; PDB 1NWK and PDB 1J6Z: representative actin monomer structures bound to ATP or ADP, respectively53,54.

Extended Data Fig. 8 Flattening causes changes in the barbed end groove that facilitate long pitch interactions with actin subunits.

a, Molecular surface representation of active Arp3 showing perspective of close up views of the barbed end grooves (BEGs) depicted in b. b, BEG of inactive (PDB 4JD2) or active Arp2 or Arp3 with engaged D-loop from actin subunit n + 1 or n. Residue numbers are for the S. pombe Arp2/3 complex. Green spheres and dashed line show distance x3, which measures the distance between the C-terminus and the front of the BEG. c, Plot of the twist angle versus distance x3 for inactive or active structures of actin, Arp2 or Arp3. Individually labeled data points are as described in Extended Data Fig. 7d.

Extended Data Fig. 9 Close up of nucleotide clefts of Arp2 and Arp3.

a, Stereo image of the nucleotide binding cleft of Arp3 showing the density of the modeled ATP phosphates and conserved catalytic residue Q160. PG: gamma phosphate. b, Stereo image of the nucleotide binding cleft of Arp2 showing the density of the modeled ATP phosphates and conserved catalytic residue Q137. c, Stereo image of Arp2 from the active structure (pink) superposed with actin (cyan) from the cryo-EM structure of AMP-PNP bound actin filaments PDB 6DJM15 showing the nucleotide and the nucleotide binding cleft.

Extended Data Fig. 10 The clamp subunits twist during activation of Arp2/3 complex.

a, Model-based surface representation of ARPC2 and ARPC4 in active or inactive structure (PDB 4JD2) with residues contacting Arp2 or Arp3 (within 3.7 Å) colored magenta or orange, respectively. The Bos taurus inactive structure (PDB 4JD2) was used for the inactive structure in this analysis because the inactive S. pombe Arp2/3 complex structure presented here is missing some sidechains at the interface with the clamp. b, Overlay of ARPC2 and ARPC4 from the active structure on ARPC2 and ARPC4 from the inactive S. pombe Arp2/3 complex structure, respectively. Residues 1-271 of ARPC2 and 4-140 of ARPC4 were used to overlay the structures.

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Supplementary Video 1

Model of Dip1-activated Arp2/3 complex on the end of the nucleated actin filament. Arp2 and Arp3 mimic actin subunits to make filament-like contacts with the first two actin subunits in the nucleated actin filament.

Supplementary Video 2

Morph showing movement of Arp2/3 complex from the inactive to active conformation. Twisting of the ‘clamp subunits’ in the Arp2/3 complex during activation shifts half of the subunits to a new activated position.

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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). https://doi.org/10.1038/s41594-020-0481-x

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