PDBsum entry 3cfv

Histone/chaperone

PDB id: 3cfv

Contents

Protein chains

393 a.a. *

18 a.a. *

14 a.a. *

Ligands

ARS ×2

Waters ×275

* Residue conservation analysis

PDB id:

3cfv

Name:

Histone/chaperone

Title:

Structural basis of the interaction of rbap46/rbap48 with histone h4

Structure:

Histone-binding protein rbbp7. Chain: b, a. Synonym: retinoblastoma-binding protein 7, rbbp-7, retinoblastoma- binding protein p46, histone acetyltransferase type b subunit 2, nucleosome-remodeling factor subunit rbap46. Engineered: yes. Histone h4 peptide. Chain: e, f. Fragment: unp residues 25 to 42.

Source:

Homo sapiens. Human. Gene: rbbp7, rbap46. Expressed in: spodoptera frugiperda. Expression_system_cell_line: sf9. Synthetic: yes. Other_details: the peptide is chemically synthesized.

Resolution:

2.60Å R-factor: 0.202 R-free: 0.257

Authors:

X.-Y.Pei,N.V.Murzina,W.Zhang,S.Mclaughlin,A.Verreault,B.F.Luisi, E.D.Laue

Key ref:

N.V.Murzina et al. (2008). Structural basis for the recognition of histone H4 by the histone-chaperone RbAp46. Structure, 16, 1077-1085. PubMed id: 18571423 DOI: 10.1016/j.str.2008.05.006

Date:

04-Mar-08 Release date: 10-Jun-08

Protein chains

Q16576  (RBBP7_HUMAN) -  Histone-binding protein RBBP7 from Homo sapiens

Seq: 425 a.a.

Struc: 393 a.a.

Protein chain

P62805  (H4_HUMAN) -  Histone H4 from Homo sapiens

Seq: 103 a.a.

Struc: 18 a.a.

Protein chain

P62805  (H4_HUMAN) -  Histone H4 from Homo sapiens

Seq: 103 a.a.

Struc: 14 a.a.

DOI no: 10.1016/j.str.2008.05.006

Structure 16:1077-1085 (2008)

PubMed id: 18571423

Structural basis for the recognition of histone H4 by the histone-chaperone RbAp46.

N.V.Murzina, X.Y.Pei, W.Zhang, M.Sparkes, J.Vicente-Garcia, J.V.Pratap, S.H.McLaughlin, T.R.Ben-Shahar, A.Verreault, B.F.Luisi, E.D.Laue.

ABSTRACT

RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed beta propeller structure and binds histone H4 in a groove formed between an N-terminal alpha helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.

Selected figure(s)

Figure 2.
Figure 2. Histone H4 Recognition by the RbAp46-Binding Pocket
(A) Electrostatic surface potential of RbAp46 contoured and color coded at −91 kT (red) and +91 kT (blue). The potential was calculated and displayed with the program PyMol (DeLano, 2002). The histone H4 peptide is shown as a stick model. The histone H4-binding pocket in RbAp46 is mainly formed by the negatively charged PP loop (which terminates in Pro-362 and Pro-363) and a hydrophobic surface on the N-terminal α helix (helix 1). The interaction of histone H4 residues (Gln-27, Lys-31, Ile-34, Arg-35, Arg-36, Leu-37, Arg-39, and Arg-40) is shown.
(B) Detailed view showing the interactions of the hydrophobic Ile-34 and Leu-37 histone H4 residues with Phe-29 and Leu-30 in helix 1 of RbAp46, as well as the positively charged Arg-36, Arg-39, and Arg-40 histone H4 residues with the backbone carbonyl groups in the PP loop and a cluster of acidic residues (Glu-356, Asp-357, and Asp-360) in RbAp46.
(C) Site-directed mutagenesis of RbAp46 in either the charged PP loop (E356Q + D357N + E359Q + D360N), the hydrophobic surface of helix 1 (L30Y), or both simultaneously all disrupt the interaction with histone H4 in pull-down experiments with GST-histone H4 1–48.
(D) In reciprocal experiments, mutation of histone H4 residues interacting with either the charged PP loop (R39V + R40N), or of residues interacting with both helix 1 and the charged PP loop (I34T + L37D + R35S) and (L37D + R39V + R40N) also disrupt the binding. In both (C) and (D), the top panel shows an autoradiogram illustrating the amount of ^35S-labeled RbAp46 pulled down in each experiment, whereas the lower panel shows a Coomassie blue-stained gel indicating the amount of either GST or GST-histone H4 (1–48) used. In each experiment, the input lane contains 30% of the ^35S-labeled RbAp46 protein used in each of the pull-down assays. The experiments were carried out in 300 mM NaCl, 20 mM Tris (pH 8.0), 5 mM DTT, and 0.1% (v/v) NP-40.

Figure 3.
Figure 3. Interaction of Histones H3 and H4 with RbAp46
(A) Analytical size-exclusion chromatography of the recombinant histone H3/H4 complex used in the biochemical experiments described in this paper. The inset panel shows a Coomassie blue-stained 4%–12% NuPAGE gel used to analyze the fractions. In the conditions used here (2 M NaCl and 20 mM HEPES [pH 7.5], on a Superdex 75 PC3.2/30 column), histones H3 and H4 are present as tetramers, but at lower ionic strengths (as used in the binding experiments) these dissociate to form a mixture of dimers and tetramers (Banks and Gloss, 2003).
(B) Pull-down of either wild-type or mutant RbAp46 by histones H3 and H4 crosslinked to DynaBeads, in the absence or presence of the N-terminal histone H4 peptide (residues 16–41). (The experiments were carried out as described in Figure 2. The positively charged lysozyme protein was also crosslinked to beads in separate experiments and was used as a negative control.)
(C) Comparison of the interactions of Ile-34, Leu-37, and Ala-38 in helix 1 of histone H4 with (i) the N-terminal helix of RbAp46 in the RbAp46/histone H4 peptide structure, (ii) α helices 2 of histone H3 and H4 in one (of the two) H3/H4 dimer in the nucleosome core particle (Davey et al., 2002; PDB code: 1KX5), and (iii) α helices 2 of histone H3 and H4 in the ASF1-histone H3/H4 complex ([English et al., 2006] and [Natsume et al., 2007]; PDB code: 2HUE). In (i), (ii), and (iii), the view is down the axis of helix 1 of histone H4. Because similar contacts are made between histones H3 and H4 in the complex with ASF1 and in both copies of histones H3 and H4 in the nucleosome core particle, it is likely that isolated histones H3 and H4 also interact with each other in a similar manner. Histone H4 is colored blue in all three structures, whereas histone H3 is yellow in the nucleosome core particle and pink in the ASF1 complex.

The above figures are reprinted from an Open Access publication published by Cell Press: Structure (2008, 16, 1077-1085) copyright 2008.

Figures were selected by an automated process.