ABSTRACT
Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 Å resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin.
HIGHLIGHTS
The ATAD2B bromodomain recognizes mono- and di-acetylated histone ligands.
Chemical shift perturbations outline the ATAD2B bromodomain acetyllysine binding pocket.
An ATAD2B bromodomain-inhibitor complex reveals important binding contacts.
An alternate splice variant in the ATAD2B bromodomain abolishes histone and inhibitor binding.
Competing Interest Statement
EpiCypher (M.R.M. and M.-C.K.) is a commercial developer of the dCypher peptide-binding platform used in this study.
Footnotes
Significant new data was added to the current manuscript draft including, results from a histone peptide array screen of the ATAD2B bromodomain with nearly 300 unique PTM histone peptides, data identifying and characterizing an ATAD2B splice variant that abolishes histone and inhibitor binding, and inclusion of a new structure of the ATAD2B bromodomain in complex with a small molecule inhibitor with improved electron density. We also expanded the ITC binding data and reanalyzed the NMR titration data.
ABBREVIATIONS
- ATAD2
- ATPase family AAA+ domain containing 2
- ATAD2B
- ATPase family AAA+ domain containing 2B
- BRD
- bromodomain
- BRPF1/3
- Bromodomain and PHD finger containing protein 1/3
- CD
- circular dichroism
- GST
- glutathione-S-transferase
- HSQC
- heteronuclear single quantum coherence
- ITC
- isothermal titration calorimetry
- NMR
- nuclear magnetic resonance
- PHD
- plant homeodomain
- PTMs
- post-translational modifications