Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

Abstract

The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Sitedirected mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligandbinding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module. K E Y W O R D S acetyllysine, ATAD2, bromodomain inhibitor, chromatin reader domain, disulfide bridge, epigenetics, histone, post-translational modification

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

“…Our results also show that the interaction of the BRPF1 bromodomain interaction with histone ligands is enthalpy-driven. A similar result was also observed for the BRPF1 bromodomain with smallmolecule ligands, and with the ATAD2 bromodomain-ligand complexes 46,47 . The enthalpy (ΔH) and entropy (ΔS) changes obtained from fitting the binding isotherm illustrate the mechanism driving the protein-ligand interaction 48 .…”

A Qualified Success: Discovery of a New Series of ATAD2 Bromodomain Inhibitors with a Novel Binding Mode Using High-Throughput Screening and Hit Qualification

“…Most constructs had similar melting temperatures, while a designed hexamutant (construct 4) was more stable and another (construct 8) was biphasic. We note in passing that the design of the hexamutant construct was partly intended to remove cysteine residues, which we speculated could influence the stability of the bromodomain, including the neighboring pair Cys1057/Cys1079, an idea that has recently been discussed by others …”

Functional Roles of Bromodomain Proteins in Cancer

“…This finding supports previous research showing the bromodomain of ATAD2 functions to recognize H4K5ac and H4K12ac modifications [ 183 , 184 ]. Interestingly, the presence or absence of a disulfide bridge located at the bottom of the bromodomain binding pocket influences ligand recognition of ATAD2, thus acetyllysine binding could be tied to the redox status of the cell [ 191 ]. In addition, it appears that the ATPase domain plays an important role in chromatin recruitment, as the bromodomain alone was not sufficient.…”