Bromodomains exhibit preferences for specific patterns of post-translational modifications on core and variant histone proteins. We examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralogue in ATAD2. We show that the ATAD2B bromodomain recognizes mono- and diacetyllysine modifications on histones H4 and H2A. A structure–function approach was used to identify key residues in the acetyllysine-binding pocket that dictate the molecular recognition process, and we examined the binding of an ATAD2 bromodomain inhibitor by ATAD2B. Our analysis demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, with many residues playing a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of function. Our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism regulating the interaction of the ATAD2B protein with chromatin.
Bromodomain proteins function as epigenetic readers that recognize acetylated histone tails to facilitate the transcription of target genes. There are approximately 60 known human bromodomains, which are divided into eight sub-families based on structural conservation. The bromodomain-containing proteins in family IV include seven members (BRPF1, BRPF2, BRPF3, BRD7, BRD9, ATAD2, and ATAD2b). The bromodomains of each of these proteins recognize and bind acetyllysine residues on histone tails protruding from the nucleosome. However, the histone marks recognized by each bromodomain protein can be very different. The BRPF1 subunit of the MOZ histone acetyltransferase (HAT) recognizes acetylated histones H2AK5ac, H4K12ac, H3K14ac, H4K8ac, and H4K5ac. While the bromodomain of BRD7, a member of the SWI/SNF complex, was shown to preferentially recognize acetylated histones H3K9ac, H3K14ac, H4K8ac, H4K12ac, and H4K16ac. The bromodomains of BRPF2 and BRPF3 have similar sequences, and function as part of the HBO1 HAT complex, but there is limited data on which histone ligands they bind. Similarly, there is little known about the histone targets of the BRD9 and ATAD2b bromodomain proteins. Interestingly, the ATAD2 bromodomain was recently shown to preferentially bind to the di-acetylated H4K5acK12ac mark found in newly synthesized histones following DNA replication. However, despite the physiological importance of the family IV bromodomains, little is known about how they function at the molecular or atomic level. In this review, we summarize our understanding of how family IV bromodomains recognize and select for acetyllysine marks and discuss the importance of acetylated histone recognition for their biological functions.
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
Reverse Cover: The cover image is based on the Structure Note Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain by Jamie C. Gay et al., DOI: https://doi.org/10.1002/prot.25636.
The ATPase Family, AAA domain‐containing protein 2 (ATAD2) bromodomain has canonical bromodomain structure, consisting of four α‐helices. ATAD2 is known to be up‐regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate cancer. ATAD2 is a co‐activator of the androgen and estrogen receptors, as well as MYC and E2F transcription factors. Recently, ATAD2 was shown to read newly synthesized diacetylated marks during DNA replication, and ATAD2 overexpression further increases ATAD2 levels, cell proliferation, and survival genes. Furthermore, up‐regulation of ATAD2 is strongly correlated with poor patient prognosis. This makes the ATAD2 bromodomain an innovative target for cancer therapeutics, and several inhibitors are currently in development. Interestingly, the ATAD2 bromodomain contains two cysteine residues near the base of the bromodomain‐binding pocket between helices αB and αC. We hypothesized that disulfide bridge formation between residues C1057 and C1079 might regulate the interaction of the ATAD2 bromodomain with its histone ligands. X‐ray crystallography confirmed formation of a disulfide bridge in the ATAD2 bromodomain. The role of the disulfide bridge in ATAD2 bromodomain stability was explored through mutagenesis, codon optimization, and expression experiments. To investigate the impact of disulfide bridge formation on histone ligand recognition by the ATAD2 bromodomain, isothermal titration calorimetry was carried out to characterize binding affinities of histone ligands using the ATAD2 bromodomain protein with and without an intact disulfide bridge. This study demonstrates the functional effect of disulfide bridge formation upon histone ligand recognition by the ATAD2 bromodomain, and presents additional structural information, which may prove essential for effective inhibitor development for the treatment of many cancers.Support or Funding InformationNIGMS R15 215GM104865, GlassStudent Summer Research Award, ACPHS Graduate Research Assistantship, EvansThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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