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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.
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