Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin

Abstract

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.

Figure 1

Structure and sequence comparisons between Arp4 and actin. (A) Sequence alignment between S. cerevisiae Arp4 (top) and actin (bottom). Identical residues are shaded in red and similar residues in yellow. The main differences between the two proteins: shortening of the DNaseI loop, Insertion I (25 residues) and Insertion II (80 residues) are highlighted by pink boxes. The secondary structure of Arp4 is displayed on the top of the alignment; residues missing in the electron density are depicted as dashed lines. (B) Crystal structure of S. cerevisiae actin (pdb: 1YAG). The four subdomains are numbered and the DNaseI loop is labelled. ATP is represented in sticks and the metal ion is displayed as a sphere. (C) Crystal structure of S. cerevisiae Arp4 (pdb: 3QB0). The two insertions are labelled and shown in pink, as is the shortened DNaseI loop. The 49 amino acids within Insertion II that are not visible in the electron density are indicated by the dashed line. ATP is represented in sticks and the metal ion is displayed as a sphere.

Figure 2

Solution structures of Arp4 and Arp8. (A) Overlay of the final averaged ab initio shape reconstruction of Arp4 derived by SAXS experiments (blue envelope) with the docked crystal structure of Arp4 (yellow). Front and side views indicate a good fit between the crystal and the solution structure. The likely position of the disordered 49 amino acids of Insertion II within the solution structure is indicated by the dashed line. (B) Overlay of the final averaged ab initio shape reconstruction of full-length Arp8 (blue envelope) and N-terminally truncated Arp8 lacking the first 244 amino acids (red envelope) derived from SAXS experiments. The crystal structure of yeast actin (pdb: 1YAG, cyan) is docked into the envelopes for comparison. Front and side views indicate that the core actin fold fits into both solution structures of Arp8. Additional density can be attributed to insertions present in Arp8 compared with actin. The large N-terminus of Arp8 (amino acids 1–244) seems to form an extended protrusion consistent with the secondary structure prediction for it to be mainly unstructured.

Figure 3

ATP binding by Arp4. (A) Stereo image of the simulated annealing difference omit map calculated for the ATP molecule and the metal ion using CNS. Electron density is clearly present between the four domains of Arp4 (yellow), indicating binding of the ATP and metal ligands (orange sticks and sphere). The map is displayed as blue mesh and contoured at 1.0 σ. (B) Detailed view of the ATP nucleotide bound by actin (pdb: 2HF4). The protein is displayed in cyan ribbon and important residues are represented as sticks and labelled. (C) Detailed view of the ATP nucleotide bound by Arp4 (in yellow). Important residues are represented as sticks and labelled. Note that Tyr 24 stacks on the ribose of ATP and Asp163 forms a hydrogen bond with Ser23 leading to tighter closure of the two nucleotide-binding loops P1 and P2. Together with His162 and helix 227–246 (in pink) the nucleotide is more strongly shielded from the solvent as compared with actin.

Figure 4

Arp4 is not capable of forming actin-like filaments. (A) Model of two intra-strand actin monomers (n and n+2) preceding each other within the actin filament (Oda et al, 2009) (two shades of cyan). Important residues for forming intra-strand contacts are highlighted in dark green and labelled. Note the extended DNaseI loop of the bottom monomer reaching into the hydrophobic groove between subdomains one and three of the following monomer. (B) Model of inter-strand interactions between two preceding actin monomers (n and n+2) with a third monomer from the second strand (n+1) of the double-stranded actin filament (Oda et al, 2009) (three shades of cyan). Important residues for forming inter-strand contacts are highlighted in dark green and labelled. Note the hydrophobic plug region extending from the n+1 monomer to contact both opposing actin monomers. (C) Overlay between actin (cyan) and Arp4 (yellow) detailing differences at the pointed end. Highlighted in dark green and labelled are actin residues important for forming contacts within the actin filament (see Figure 4A). In pink are changes present in Arp4 that render those contacts largely impossible (Insertion I and shortened DNaseI loop). (D) View of the ‘backside’ of actin (cyan) and Arp4 (yellow). The hydrophobic plug region of actin is coloured in dark green and labelled. Insertion II (in pink; dashed line represents the unstructured 49 residues of Insertion II) expands this region in Arp4 and Trp315 (represented in sticks and labelled) caps α-helix 110–115 in actin, rendering important inter-strand contacts within the actin filament impossible (see Figure 4B).

Figure 5

Arp4 inhibits F-actin assembly. (A) Pyrene assays of actin polymerization. In all, 4 μM Mg²⁺-ATP-actin was polymerized by addition of KMEI buffer and the Arp4 concentrations indicated, and the increase in pyrene fluorescence was detected over time. (B) Normalized polymerization rates derived from three independent experiments equivalent to (A) show a dose-dependent inhibition of polymerization. (C) Steady-state F-actin fluorescence from experiments equivalent to (A) indicate that Arp4 does not sequester monomers. (D) Co-sedimentation assays of 5 μM Mg²⁺-ATP-actin polymerized in the presence of the Arp4 concentrations indicated shows that Arp4 neither sequesters monomers nor binds to F-actin. (E) In vitro TIRF microscopy of single actin filaments growing in the presence and absence of Arp4 at time points 0, 100 and 200 s. In all, 1.3 μM Mg²⁺-ATP-G-actin was polymerized in TIRF buffer and the elongation rates and numbers of filaments were analysed. Addition of Arp4 markedly decreases the number of growing filaments and has a slight effect on the elongation rate as well.

Figure 6

Effects of Arp4 on the G-/F-actin equilibrium. (A) Critical concentration plot of F-actin in the presence and absence of Arp4 and CapZ. F-actin at different concentrations, either with free barbed ends or with CapZ-capped barbed ends was incubated in the presence and absence of 20 μM Arp4. Arp4 only increases the critical concentration of capped filaments. (B) Time course of the effects of Arp4 on the G-/F-actin equilibrium. Pyrene fluorescence of F-actin was detected for 200 s. Subsequently, KMEI buffer alone or supplemented with Arp4 or LatA was added, and the decrease in fluorescence was followed for 10 000 s. The smaller timescale highlights fast, instant depolymerization upon addition of Arp4 (right). (C) Effect of Arp4 on the G-/F-actin equilibrium when barbed ends are capped. The experiments were performed as in (B) with the exception that CapZ was added. The smaller timescale illustrates that rapid Arp4-mediated F-actin disassembly is abolished when barbed ends were capped (right). (D) Effect of Arp4 on the F-/G-actin equilibrium in the absence of ATP. The experiments were performed as in (B). Buffers contained ADP instead of ATP, residual ATP was removed by adding hexokinase and glucose before the experiments. The smaller timescale highlights immediate and complete depolymerization of ADP-actin by Arp4 (right). (E) ADP-actin assembly assay. In all, 10 μM Mg²⁺-ADP-actin was polymerized in the presence of the Arp4 concentrations indicated. (F) Normalized polymerization rates derived from (E) and Figure 5A are shown for comparison. The inhibition of actin assembly by Arp4 is much more efficient for ADP-actin. (G) Addition of ATP to Arp4-inhibited ADP-Mg²⁺-actin polymerization restored actin assembly.

Figure 7

Arp8 does not inhibit actin assembly but sequesters actin monomers. (A) Pyrene assays of actin polymerization. In all, 4 μM Mg²⁺-ATP-actin was polymerized by addition of KMEI buffer and the Arp8 concentrations indicated, and the increase in pyrene fluorescence was detected over time. (B) Normalized polymerization rates derived from three independent measurements equivalent to (A) show no inhibition of polymerization by Arp8. (C) Number of filaments and barbed end elongation rates in the presence and absence of Arp8 determined by TIRF microscopy as described in Figure 5E are not influenced by Arp8. (D) Steady-state F-actin fluorescence measurements indicate that Arp8 sequesters monomers. (E) Co-sedimentation assays of 5 μM actin polymerized in the presence of the Arp8 concentrations indicated corroborate that Arp8 sequesters monomers. (F) Critical concentration plot of F-actin in the presence and absence of Arp8 and CapZ. Conditions are as in Figure 6A. Note that the change in C_Crit does not depend on the presence of CapZ. C_Crit(free) and C_Crit(capped) are both shifted to 1.3 μM corresponding to a K_D of the Arp8–G-actin interaction of ∼16.5 μM. (G) Arp8 depolymerizes F-actin with very slow kinetics. Conditions are as in Figure 6B and D. Note that removal of ATP from the reaction mixture led to a loss of the pronounced lag-phase of Arp8-mediated F-actin disassembly. (H) ADP-actin assembly assay. In all, 10 μM Mg²⁺-ADP-actin was polymerized in the presence of the Arp8 concentrations indicated. The amount of F-actin was reduced with increasing amounts of Arp8.

Figure 8

Arp4 and Arp8 synergistically sequester G-actin. (A) Pyrene assays of actin polymerization. In all, 4 μM Mg²⁺-ATP-actin was polymerized by addition of KMEI buffer in the presence of 6 μM Arp4 and the Arp8 concentrations indicated. (B) Arp8 enhances Arp4-mediated inhibition of polymerization. Polymerization rates were obtained from (A) and normalized to the slope of the assembly of 4 μM Mg²⁺-ATP-actin in presence of 6 μM Arp4. (C) Single filament elongation rates and numbers of filaments in the presence and absence of 5 μM Arp4 and 20 μM Arp8 obtained by TIRF microscopy as described in Figure 5E indicate that Arp8 increases the effect of Arp4. (D) Steady-state F-actin fluorescence experiments indicate that Arp4 increases Arp8-mediated sequestering of monomers. (E) Co-sedimentation assays of 5 μM actin polymerized in the presence of the Arp4 and Arp8 concentrations indicated. The sequestration effect of Arp8 is more pronounced in the presence of Arp4 (compare with Figure 7E). (F) Combined effects of Arp4 and Arp8 on the F-/G-actin equilibrium suggest an additive and sequential effect of fast depolymerization by Arp4 and slow depolymerization by Arp8. Experiments were performed as in Figure 6B.