Structural basis of histone H4 recognition by p55

Abstract

p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a beta-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.

Figure 1.

Crystal structures of p55 and a complex with histone H4 peptide. (A) Ribbon diagram of the p55 crystal structure. p55 contains WD40 repeats forming a seven-bladed β-propeller structure (yellow) with an additional α-helix at the N terminus (blue). Disordered loops were connected and are shown in dashed lines. (B) Ribbon diagram of the p55 bound with histone H4 peptide. Histone H4 (₃₁KPAIRRLARRG₄₁, shown in red) is bound at the side of the β-propeller structure and between the N-terminal α-helix (blue) and the binding loop coming from the seventh blade of the structure.

Figure 2.

p55 recongizes histone H4 via the binding pocket. (a) Electrostatic surface potential representation of the binding pocket with the histone H4 peptide (shown in stick model). The binding pocket is characterized by the high negative charge on the binding loop and the hydrophobic surface on the N-terminal α-helix. (b) Detailed interactions between histone H4 and the p55-binding pocket. Arg39 of histone H4 is inserted in the binding pocket and interacts with the carboxylates of Asp362 and Asp365 in the binding pocket. Leu37 and Ile39 of histone H4 interact with the hydrophobic patch formed on the surface of the N-terminal α-helix. (c) Arg39 of histone H4 forms a hydrogen bond network with carbonyl oxgens of the backbone at the binding pocket. (d) Full-length H4 was superimposed on the p55–H4 complex. The histone fold of histone H4 must be altered to allow p55 binding.

Figure 3.

Histone-binding properties of p55. (A) Purified wild-type and mutant p55. Wild-type and mutant p55 were used as analytes at the concentrations of 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1 μM, and 2 μM on CM5 chips immobilized with full-length histone H4 (B–D); full-length mutant histone H4, H4R39A (E); histone H3-H4 tetramer (F–H); mutant tetramer, H3-H4R39A (I); and full-length histone H3 (J–L) with 520, 430, 177, 180, and 493 RU, respectively.

Figure 4.

HAT activities of human Hat1 complex. (A) Purified hHat1 subunit alone (N-terminal His-tagged), wild-type human Hat1 complex (His tag on RbAp48), and Hat1 complexes containing mutant RbAp48. (B) Mutations in the binding pocket of RbAp48 decrease the HAT activity. (A,C) Equal amounts of human HAT complexes and full-length H4 were used for HAT assay.