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.