Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex

Abstract

Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.

Fig. 1.

Crystal structure and nucleotide binding of yArp8CTD. (A) Overall structure of yArp8CTD. The color scheme used matches that in SI Appendix, Fig. S1, which summarizes the secondary structure. The bound ADP is shown in magenta. (B) Isothermal titration calorimetry data for ATP binding to yArp8CTD. (C) The ADP-binding site in yArp8CTD. ADP and selected residues are shown as sticks, with the Fo–Fc difference density superimposed at the 3σ contour level. (D) ATP-binding site of yeast actin (PDB ID code 1YAG; magenta), with corresponding residues of yArp8CTD (yellow) superimposed.

Fig. 2.

yArp8CTD interacts with histones. (A) Pull-down experiments for GST-yArp8CTD with histones H2A, H2B, H3, H4, and H3/H4. Each pair of lanes corresponds to GST control (Left) and GST-yArp8CTD (Right). (B) Decamer peptide arrays for histone H3 (Upper) and H4 (Lower) probed with yArp8CTD. For the H3 array, row L1 corresponds to 20 sequential peptides from residues 1–10 (A), then residues 2–11 (B), through to residues 20–29 (T). Each row down presents the next 20 peptides. The H4 array follows a similar pattern beginning at row L8 (A). (C) Data from B overlaid on the nucleosome structure. Histones H3 (yellow) and H4 (green) are highlighted in red to indicate positive peptides from the array data. (D) Pull-down experiments with GST-tagged inserts 2A, 3A, and 3B with H3/H4 tetramers. Lanes are as follows: GST, GST control; Arp8, GST-yArp8CTD plus H3/H4; 2A, GST insert 2A plus H3/H4; 3A, GST insert 3A plus H3/H4; 3B, GST insert 3B plus H3/H4; and H3/H4, H3/H4 alone.

Fig. 3.

EM 3D image reconstruction of the yArp8CTD–nucleosome complex. Bottom view (A) and side view (B) showing yArp8CTDs (magenta) binding to each face of the nucleosome (blue). The arrow indicates H3/H4 subunits that do not fit into the density. The red ellipse shows additional density that is unaccounted for by the crystal structures.

Fig. 4.

Purification and analysis of full-length his-tagged yArp8 protein. (Upper) Elution profile from heparin-Sepharose column and SDS gel of wash (W) and eluate (E) fractions. (Lower) Analysis after gel filtration by MALS of protein fractions that passed through the column (wash, Left) or were bound and eluted with a salt gradient (elution, Right). MALS analysis of unbound protein gave a molecular mass estimate of 94.6 kDa, corresponding to monomers (100.2 kDa) of yArp8. Bound protein contained a more complex mixture, with MALS analysis showing a major peak at 231 kDa (dimer) and minor peaks at 108 kDa (monomer) and 446 kDa.

Fig. 5.

Negative-stain EM reconstruction of the yArp8 dimer. (A) View showing the channel flanked by the yArp8CTDs. The three inserts—2A (blue), 3A (green), and 3B (red)—line the channel walls. (B) “End” view; the yArp8CTD crystal structure closely fits the density, with the exception of insert 3A (green), which sits above and adjacent to unaccounted-for density (red ellipse). (C) Overlay of yArp8 dimer (cyan) with yArp8CTD–nucleosome (gray). (D) Enlarged view of density corresponding to yArp8CTD in the two reconstructions, showing the rotation required to open the cleft to accommodate a nucleosome. For clarity, density corresponding to the nucleosome is omitted.

Fig. 6.

Superdex S200 gel filtration of a molar excess of H3/H4 tetramers mixed with either monomeric or dimeric yArp8. Fractions from each gel filtration run were analyzed on SDS/PAGE gels, as shown below the trace.