The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p

Abstract

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

Fig. 1. Sequence of the Gcn5p bromodomain used for crystallization and for NMR spectroscopy. Highly and absolutely conserved residues in the bromodomain are marked by one and two asterisks, respectively. Amino acids encoded by the vector are in lower case. Residues directly contacting the N-acetyl lysine in the peptide are indicated by closed circles and residues contacting other parts of the peptide by open circles. The nomenclature for the secondary structure follows that of Dhalluin et al. (1999). α_Z′ is a short α-helix that is present in all bromodomain structures but was previously unnamed. α- and 3₁₀-helices are indicated by dark grey and light grey boxes, respectively.

Fig. 2. The Gcn5p bromodomain binds specifically to an H4 peptide containing N-acetyl lysine. Superimposed (¹⁵N, ¹H) HSQC spectra of free bromodomain and bromodomain plus unacetylated peptide (left) and free bromodomain and bromodomain plus acetylated peptide (right). In both cases, the spectrum for the free bromodomain is shown in black and that complexed with the peptide in red.

Fig. 3. Histogram of chemical shift changes induced on addition of H4 peptide acetylated at Lys16. ¹H and ¹⁵N changes are denoted by filled and open bars, respectively.

Fig. 4. Overall structure and peptide binding. (A) Ribbon diagram showing the bundle of four main α-helices, Z, A, B and C, with the Nζ-acetyl lysine side chain of the peptide bound in a deep slot at the top of the bundle. The ribbon is coloured from green (N-terminus) to gold (C-terminus). (B) ‘Top’ view of the molecular surface coloured by hydrophobic potential (dark green marks favourable regions for hydrophobic interaction; grey, less favourable; light green, intermediate). The five visible residues of the peptide fit into a shallow groove, with the acetyl lysine side chain in a deep slot. The hydrophobic surface at the base of the pocket formed by Phe352 and Val356, as well as that lining the side of the pocket formed by Pro351, Val 356, Tyr364 and Tyr413, are not easily visible in this view. (C) The same view as (B), showing the underlying helices coloured as in (A). (D) Mapping of backbone NH chemical shift changes onto residues 329–438 of the peptide backbone of the Gcn5p bromodomain in the same orientation as (B) and (C). ¹H and ¹⁵N changes are indicated in red and blue, respectively. The colours are merged so that the shade reflects the relative contributions of the two shift changes and the intensity reflects the magnitude of the changes. Residues for which no backbone amide groups were assigned are shown in yellow (see text).

Fig. 5. Details of the peptide binding site. (A) Schematic view of interactions, with water molecules represented as W. (B) The N-acetyl lysine slot showing the ring of water molecules around the acetyl group at the base of the slot, and the hydrophobic walls left and right. (C) The binding groove for the (K + 2) and (K + 3) peptide residues lies across the 405–408 loop between α_B and α_C. His(K + 2) packs against Phe367, and Arg(K + 3) forms hydrogen bonds back to the protein backbone. The peptide backbone forms four hydrogen bonds to the protein, three of them via water molecules.

Fig. 6. The Nζ-acetyl lysine binding site, showing electron density from the final 2mF_o – DF_c map. Nitrogen atoms are black and oxygen atoms are grey. The acetyl group is surrounded by a ring of water molecules (grey balls) at the base of the slot, and the carbonyl oxygen of the acetyl forms a hydrogen bond to the side chain of Asn407. An unacetylated lysine could not form these hydrogen bonds, and would introduce an unpaired charge into the slot.