Chromatin-modifying complex component Nurf55/p55 associates with histones H3 and H4 and polycomb repressive complex 2 subunit Su(z)12 through partially overlapping binding sites

Abstract

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

FIGURE 1.

Overview of the Nurf55-H4 peptide structure. A, front and top views (turned by 90° around a horizontal axis) of the Nurf55-H4 peptide structure. Nurf55 is depicted in marine, and the H4 peptide is shown in yellow. Numbers in the lower left panel indicate the blades of the β-propeller. B, electrostatic potential mapped on the surface of Nurf55. Acidic patches are shown in red, neutral patches in white, and basic patches in blue (overall range from −30 to +30 kT). C, sequence conservation mapped on the surface of Nurf55. Completely conserved residues are indicated in violet, residues >80% conserved in purple, and residues >60% conserved in light blue.

FIGURE 2.

Overview of the Nurf55-peptide interaction. A, stereo view of polar interactions between Nurf55 and the H4 peptide. Nurf55 and the peptide are depicted as blue and yellow ribbons, respectively. Interacting residues enclosed by a 2F_o − F_c electron density map at 1.0σ are depicted in stick representation. B, schematic representation of the Nurf55-peptide interactions. The three helices and blade 6 forming the binding pocket and blade 7 are depicted in blue, the connecting residues between blades 7 and 6 as a dashed blue line, and the H4 peptide in orange. Hydrophobic interactions are drawn in black, hydrogen bonds in red, and water-mediated hydrogen bonds as dashed red lines. The hydrogen bond network shown in C is indicated with green lines. C, close-up view of the hydrogen bond network between the NH₂ group of Arg-39, the carboxamide of Gln-358, and the main chain carbonyls of Asp-362 and Gly-366. The network is indicated with dashed green lines.

FIGURE 3.

Interaction of Nurf55 with histones H3 and H4. A, secondary structure elements of histones H3 and H4 from D. melanogaster. Peptides H3_1–28, H3_1–15, H3_6–20, H3_13–28, and H4_26–45 used for ITC measurements and pulldown assays are indicated. Peptides containing trimethylated Lys-4, Lys-9, and Lys-27 residues as indicated by asterisks were tested for Nurf55 binding. The sequences of both peptides are given. B, side view of the Nurf55 β-propeller. Residues mutated in the H4-binding groove are depicted. C, top view of Nurf55 with the residues mutated in the H3 tail-binding site indicated. D, site-directed Nurf55 mutants of the H3-binding site (E235Q/D252K/E279Q; NmH3) and the H4-binding site (D362A/D365A; NmH4). Wild-type Nurf55 or Nurf55 mutants E235Q/D252K/E279Q and D362A/D365A were pulled down by histone H3/H4 dimers cross-linked to Dynabeads. Lysozyme was also cross-linked to beads and was used as a negative control. In each experiment, the input lane (i) contains one-tenth of the protein used in each of the pulldown assays, and lane b corresponds to the proteins pulled down. Lane M, molecular mass markers.

FIGURE 4.

Interaction of Nurf55 with the PRC2 component Su(z)12. A, pulldown experiments of wild-type Nurf55 and mutants D362A/D365A and L35S/F372S/I373S with FLAG-tagged full-length Su(z)12. B, Western blot of cell lysates with anti-Nurf55 antibodies demonstrates that wild-type and mutant Nurf55 proteins were expressed at comparable levels.

FIGURE 5.

Interaction of Nurf55 with the N terminus of Su(z)12. A, full-length Su(z)12 and fragments Su(z)12-1–9 were expressed in E. coli as N-terminal GST fusion proteins. Constructs were designed to preserve the predicted zinc finger (residues 411–434), predicted secondary structure elements, and the VEFS homology box (residues 527–603) (27). The results shown in B are summarized under Interaction with Nurf55. B, purification of Su(z)12 fragments using glutathione base affinity purification (upper panel) and proteins cleaved from the glutathione beads and analyzed by Western blotting with anti-Nurf55 antibodies (lower panel). Only full-length Su(z)12 and the Su(z)12-1 fragment (residues 1–100) bound Nurf55. The low signal of full-length Su(z)12 compared with bacterially expressed Su(z)12-1 presumably results from partial aggregation of this protein. IB, immunoblot. C, fixed amounts of Nurf55 in the absence or presence of increasing concentrations of Su(z)12-1 (0.5-, 1-, 10-, and 50-fold excess compared with Nurf55 (mol/mol)) were pulled down by peptide H4_26–45 fixed to Dynabeads. In each experiment, the input lane (i) contains one-tenth of the protein used in each of the pulldown assays, and lane b corresponds to the bound material. D, alignment of residues 51–100 of Drosophila Su(z)12 with the corresponding regions in Xenopus laevis and human orthologs. Predicted secondary structure elements in Su(z)12 are depicted. Helix 2 of Su(z)12 contains a similar pattern of conserved arginine residues (blue) and hydrophobic residues (red) as H4 helix 1.