Solution structure and acetyl-lysine binding activity of the GCN5 bromodomain

The solution structure of the bromodomain from the human transcriptional co activator GCN5 has been determined using NMR methods. The structure has a l eft-handed four-helix bundle topology, with two short additional helices in a long connecting loop. A hydrophobic groove and deep hydrophobic cavity a re 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 poc ket with modest affinity (K-D approximate to 0.9 mM); no comparable binding occurs with unacetylated peptides. The GCN5 bromodomain binds the small li gands N-omega-acetylhistamine and N-methylacetamide, confirming specificity for the alkyl acetamide moiety and showing that the primary element of rec ognition is simply the sterically unhindered terminal acetamide moiety of a n acetylated lysine residue. Additional experiments show that binding is en hanced if the acetyl-lysine residue occurs within the context of a basic pe ptide and is inhibited by the presence of near;by acidic residues and by th e carboxyl group of the free acetyl-lysine amino acid. The binding of the G CN5 bromodomain to acetylated peptides appears to have little additional se quence dependence, although weak interactions with other regions of the pep tide are implicated by the binding data. Discrimination between ligands of positive and negative charge is attributed to the presence of several acidi c 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 b romodomains might display differences in substrate selectivity and specific ity as well as differences in function in vivo. (C) 2000 Academic Press.