Molecular Basis of Histone Acetyl-lysine Recognition by the BRPF1 Bromodomain

In humans, translocation of the monocytic leukemia zinc-finger (MOZ) histone acetyltransferase (HAT) protein is associated with a subtype of acute myeloid leukemia (AML) with a particularly poor outcome. MOZ forms tetrameric complexes with ING5 (inhibitor of growth 5), hEAF6 (human Esa1-associated factor-6-ortholog), and the bromodomain-PHD finger protein 1 (BRPF1). BRPF1 contains a unique combination of domains typically found in chromatin-associated factors, which includes PHD fingers, a bromodomain and a PWWP (Pro-Trp-Trp-Pro) domain. Bromodomains are evolutionary conserved structural motifs generally known to recognize acetylated lysine on the histone tail. Previous studies revealed that the BRPF1 bromodomain preferentially binds to the H2AK5ac, H4K12ac and H3K14ac histone peptides. However, the molecular mechanism driving recognition of the acetylated histone tails by the BRPF1 bromodomain has not been elucidated. In this study we used X-ray crystallography to determine the structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptide ligands. These structures revealed that a network of H-bond and hydrophobic contacts coordinate the acetyllysine moiety and make specific contacts to flanking residues in the histone tail. We also identified two ordered water molecules in the binding pocket, which mediate coordination of the acetyllysine moiety. To gain a better understanding of the molecular mechanism driving specificity, we also carried out site-directed mutagenesis in combination with isothermal titration calorimetry (ITC) studies. These studies indicated that the two most important contacts for ligand coordination include the highly conserved N83 residue, which contacts the acetyllysine directly via H-bond, as well as F89, which creates a critical hydrophobic contact with the histone tail and shapes the bromodomain binding pocket wall. Circular dichroism (CD) spectroscopy studies performed on each of the wild-type and mutant proteins demonstrated that the mutations introduced into the BRPF1 bromodomain did not cause any significant change in the overall structure, or unfolding of the mutant proteins as evinced by the observed less than 6% change in &agr;-helical content. The work presented here is important for unraveling the role of the BRPF1 bromodomain in modulating the genomic binding targets of the MOZ HAT, and its potential as a therapeutic target in MOZ-related leukemia's.