The solution structure of the bromodomain from the human transcriptional coactivator GCN5 has been determined using NMR methods. The structure has a left-handed four-helix bundle topology, with two short additional helices in a long connecting loop. A hydrophobic groove and deep hydrophobic cavity are formed by loops at one end of the molecule. NMR binding experiments show that the cavity forms a specific binding pocket for the acetamide moiety. Peptides containing an N(epsilon)-acetylated lysine residue bind in this pocket with modest affinity (K(D) approximately 0.9 mM); no comparable binding occurs with unacetylated peptides. The GCN5 bromodomain binds the small ligands N(omega)-acetylhistamine and N-methylacetamide, confirming specificity for the alkyl acetamide moiety and showing that the primary element of recognition is simply the sterically unhindered terminal acetamide moiety of an acetylated lysine residue. Additional experiments show that binding is enhanced if the acetyl-lysine residue occurs within the context of a basic peptide and is inhibited by the presence of nearby acidic residues and by the carboxyl group of the free acetyl-lysine amino acid. The binding of the GCN5 bromodomain to acetylated peptides appears to have little additional sequence dependence, although weak interactions with other regions of the peptide are implicated by the binding data. Discrimination between ligands of positive and negative charge is attributed to the presence of several acidic residues located on the loops that form the sides of the binding pocket. Unlike the residues forming the acetamide binding cavity, these acidic side-chains are not conserved in other bromodomain sequences, suggesting that bromodomains might display differences in substrate selectivity and specificity as well as differences in function in vivo.
Copyright 2000 Academic Press.