 |
PDBsum entry 3cfs
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Histone/chaperone
|
PDB id
|
|
|
|
3cfs
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structural basis for the recognition of histone h4 by the histone-Chaperone rbap46.
|
|
|
Authors
|
|
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.
|
|
|
Ref.
|
|
Structure, 2008,
16,
1077-1085.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
