Crystal structure of a nuclear actin ternary complex

Abstract

Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus.

Keywords: Arp4; chromatin remodeling; crystal structure; nuclear actin.

Fig. S1.

SDS/PAGE analysis of G-actin and the N-actin complex. G-actin and the N-actin complex were purified close to homogeneity through multiple steps of chromatography.

Fig. 1.

Two different views of the overall structure of the actin–Arp4–HSA complex. Actin, orange; Arp4, green; HSA domain, magenta; the “hydrophobic plug” of actin, blue; the two insertions (In1 and In2) of Arp4, red. The N and C termini of the HSA domain are labeled with black dots. The four subdomains of actin are labeled D1, D2, D3, and D4.

Fig. S2.

Interactions with Arp4 prevent N-actin from polymerization. (A) Arp4 mainly interacts with N-actin at subdomain D3. N-actin and Arp4 are colored as in Fig. 1. Glu244 of Arp4 forms two salt bridge interactions with Arg290 of actin. Arg212 and Lys213 of the In1 of Arp4 form H bonds with Ala319 and Ser323 of actin, respectively. Ile64 and Ser61 of Arp4 pack against the main chain of the “hydrophobic plug” of actin. (B) Supposition of the actin–Arp4 complex with a F-actin model (PDB ID code 3MFP) (29). The actin protomers along the F-actin model are labeled N-1, N, and N+1, and colored gray, cyan, and light blue, respectively. The alignment was done on actin N. Arp4 clashes with the actin protomers located at the adjacent positions (N-1 or N+1) across the two-stranded filament, which provides a mechanism for preventing N-actin from polymerization.

Fig. 2.

Interactions between the HSA domain and actin/Arp4. (A) Superposition of the actin/Arp4 module with the Arp7/9 module. Arp7, pink; Arp9, blue; HSA domain of Snf2, gray. Arp4 and Arp7 were structurally aligned. (B) Multiple sequence alignments of the HSA domain. The residues involved in binding to Arp4 and actin are shown in bold (ScSwr1). The hydrophobic residues implicated in binding to N-actin and ARPs are highlighted in yellow. Dm, Drosophila melanogaster; Hs: Homo sapiens; Sc, S. cerevisiae. (C) Comparison of the HSA^Swr1–Arp4 interaction with the HSA^Snf2–Arp7 interaction. The structures of Arp4 and Arp7 are aligned. Three key hydrophobic residues of HSA^Swr1 (L355, M362, and F366) are labeled, corresponding to R604, S611, and Q615 of HSA^Snf2, respectively. (D) Comparison of the interactions around the C termini of the HSA domains. The structures of N-actin and Arp9 are aligned. The C terminus of HSA^Swr1 binds to the barbed end of N-actin and contacts multiple hydrophobic residues, including F169 (corresponding to Y169 in human β-actin), F375, and L346. Two key residues of HSA^Swr1 (V380 and I384) are labeled. Similar hydrophobic interactions were also observed at the HSA^Snf2–Arp9 interface. (E) The coimmunoprecipitation of endogenous Arp4 with Flag-tagged actin in HEK293 cells. (F) Quantification of the normalized amounts of Arp4 coimmunoprecipitated with WT actin and three mutant actins. Error bars show the SEM of at least five independent experiments. *P < 0.01.

Fig. S3.

Crystal packing environments of the N and C termini of the HSA^Swr1 helix. (A) Crystal packing environment of the N-terminal end of the HSA^Swr1 helix. Arp4 and the HSA helix within one asymmetry unit are colored as in Fig. 1A, and the nearby molecules in the crystal lattice were colored gray. (B) Crystal packing environment of the C-terminal end of the HSA^Swr1 domain. Both the N- and C-terminal ends of the HSA^Swr1 domain are free of crystal packing interactions.

Fig. S4.

Cellular localization of the WT and three mutant β-actins. Live cell images were obtained with EGFP-actin by using HEK293 cells (left column). Fixed cell images were obtained by using HeLa cells with antibody against the EGFP tag of actin, and nuclei were stained with DAPI (right three columns). WT actin formed filaments and puncta in both cell types, and distributed predominantly in the cytoplasm. In sharp contrast to the WT actin, all three mutants lost the ability to polymerize and showed diffused distribution, as indicated by the absence of filamentous structure/punctum. The G168D/Y169D mutant actin showed strong accumulation inside the nucleus in HeLa cells, which may be due to the perturbation of the nucleus-exporting pathway.

Fig. 3.

Structural features of N-actin complexed with Arp4. (A) Superposition of N-actin (orange), G-actin [cyan; Protein Data Bank (PDB) ID code 1YAG], and an actin protomer in F-actin (gray, PDB ID code 3MFP). The structural alignments were constructed on the major domain D1–D2. Upon polymerization, the two major domains of G-actin show a counterclockwise rotation of ∼13° and become flat in F-actin, whereas it is additionally twisted by a clockwise rotation of ∼6° in the N-actin complex. (B) Conformational changes in the major-domain cleft of N-actin around the latrunculin-binding pocket. N-actin was structurally aligned with G-actin (PDB ID code 1ESV). Latrunculin (Lat), in a ball-and-stick model, is surrounded by hydrophobic contacts from L16, P32, and I34 in D2 subdomain of G-actin. The arrows indicated the movement of α5 and α6. (C) Superposition of the structures of N-actin and G-actin bound to profilin (blue; PDB ID code 2BTF). The HSA helix bound to the hydrophobic cleft of N-actin clashes with profilin. (D) Superposition of the structure of N-actin with the electron density map (contour level σ = 1) of the nucleotide-binding pocket. The ADP bound by G-actin (PDB ID code 1YAG) is indicated with a stick model. No electron density at the position of ADP was found in the crystal structure of the N-actin complex. (E) Time-course of the nucleotide exchange by G-actin (red curve) and the N-actin complex (black curve). Conditions: 3 μM G-actin or N-actin complex was mixed with 50 μM ε-ATP in buffer (2 mM Tris⋅HCl, pH 7.5 and 50 mM KCl).

Fig. S5.

The twisted pointed end of N-actin disfavors the binding of tropomodulin and thymosin-β4. (A) Superposition of N-actin (orange) with G-actin (cyan) in complex with tropomodulin (light blue, PDB ID code 4PKG) (32). The actin-binding site 1 (ABS1) of tropomodulin binds across the major-domain cleft of G-actin, connecting subdomain D2 and the α6 helix of subdomain D4. In N-actin, the major-domain cleft is twisted ∼5° relative to the tropomodulin-bound G-actin, and the α6 helix is partially melted. These structural changes would disfavor the binding of tropomodulin at the pointed end of N-actin. (B) Superposition of N-actin with G-actin in complex with thymosin-β4 (blue, PDB ID code 1T44) (33). The C-terminal helix of thymosin-β4 binds across the major-domain cleft between subdomain D2 and D4 in G-actin. Because of the twisting of the pointed end of N-actin, Leu35 and Pro36 of thymosin-β4 clash with multiple sites of the α6 helix, which would inhibit the binding of thymosin-β4 to N-actin.

Fig. S6.

Binding of latrunculin to G-actin and the N-actin complex analyzed by BLI. (A) Binding of latrunculin to G-actin monitored by the super streptavidin biosensor using BLI. G-actin at a ∼2 μM concentration was labeled with biotin and bound to streptavidin-coated sensors. After washing away loosely bound materials, association was initiated by dipping the G-actin conjugated sensors in the buffer containing latrunculin at different concentrations and was monitored at real time for 120 s. Dissociation was initiated by dipping the same sensors into buffers without latrunculin and was monitored similarly. Individual graphs with indicated concentration of latrunculin were grouped together and showed. (B) Interaction of latrunculin with the N-actin complex monitored as in A. No association or dissociation of latrunculin was detected in the presence of the N-actin complex. (C) BLI analysis of the binding of latrunculin to G-actin (black) or the N-actin complex (red). Fitting the binding curve of latrunculin to G-actin yielded the K_d ∼ 1 μM, whereas no binding to the N-actin complex was observed.

Fig. S7.

Structural superposition of N-actin and cytoplasmic G-actin bound to cofilin (blue, PDB ID code 3DAW; ref. 38). Interactions with the HSA domain are incompatible with the binding of cofilin to N-actin.

Fig. S8.

Interactions between profilin and the G-actin/N-actin complex. Five micromolar GST-tagged profilin was immobilized GST-resin and mixed with 10 μM G-actin or the N-actin complex. After washing, the bound materials were eluted and analyzed with SDS/PAGE. The HSA domain polypeptide (<10 kDa) ran out of the gel. G-actin was notably bound to profilin (lane 4), whereas little N-actin was retained by profilin (lane 7). The faint actin band in lane 7 suggested profilin competed with Arp4/HSA in binding to N-actin and led to partial disassembly of the N-actin complex, as indicated by the absence of Arp4 in the elution fraction.

Fig. S9.

Structural analysis of the ATP-binding pocket of Arp4 and N-actin. (A) Superposition of the structure of the N-actin ternary complex with the electron density map at the nucleotide-binding pocket of Arp4. ATP and its contacting residues are shown as sticks, and Mg as a yellow sphere. The contour level of the map is set with σ = 1. (B) Crystal-packing environment of the ATP-binding pocket of N-actin. The Ser155-Thr160 loop, which is incompatible with the binding of nucleotide to N-actin, is colored blue, and the surrounding molecules in the crystals were colored gray. The Ser155-Thr160 loop is buried inside the major domain cleft and free of crystal packing contact.