RESUMO
The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Ligação a DNA/química , Histonas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acetilação , Proteínas de Ligação a DNA/metabolismo , Código das Histonas , Histonas/química , Humanos , Ligação Proteica , Domínios ProteicosRESUMO
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). Site-directed 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 ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Adenosina Trifosfatases/química , Cisteína/química , Proteínas de Ligação a DNA/química , Dissulfetos/química , Domínios Proteicos , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Ligantes , Lisina/química , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Solubilidade , TermodinâmicaRESUMO
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.