AAA Domain-containing Protein Research Articles

Abstract PR002: Insights into the histone code recognition by the ATAD2B bromodomain

Abstract The ATPase family, AAA domain-containing protein 2 (ATAD2/ANCCA), is an AAA nuclear co-regulator protein containing two ATPase domains and a bromodomain. Its closely related paralog, ATAD2B (KIAA1240), also contains two ATPase domains and a bromodomain. The AAA ATPase domains are broadly associated with ATP-driven molecular remodeling reactions while the bromodomains are evolutionarily conserved, chromatin ‘reader’ domains known to regulate gene expression by binding to acetyllysine on histones. Extensive studies on ATAD2 discuss its upregulation and correlation with poor prognosis in various cancers. Despite sharing high structural and sequence similarity with ATAD2, ATAD2B remains a poorly understood paralog. While previous immunostaining studies detected high expression levels of ATAD2B in breast carcinoma, there is minimal information on its role in oncogenesis. Previously, we have shown that the ATAD2B BRD can recognize mono- and di-acetyllysine modifications on N-terminal tails of histones H4 and H2A. However, since initiation and progression of cancer are characterized by multivalent histone marks, our goal is to understand how crosstalk between combinatorial histone modifications affects the acetyllysine recognition activity of the ATAD2B BRD. We hypothesize that the bromodomain recognizes the acetyllysine mark on multivalent histones and interacts with the chromatin and the ATPase domain. We expect that our results will explain the function of the ATAD2B bromodomain in the context of the overall role of ATAD2B in normal biological and cellular processes. Since bromodomains are excellent drug targets, blocking their activity will also suggest how to disrupt their possible role in oncogenesis. In this study, we have combined various innovative and interdisciplinary approaches ranging from biochemistry and structural biology to functional genomics and cellular biology to gain insights into how crosstalk between different histone H4 PTMs can modulate the chromatin ‘reader’ activity of the ATAD2B BRD. Binding affinities obtained from ITC indicate that the ATAD2B BRD can recognize the acetyllysine mark amongst multiple histone H4 PTMs. Most of these adjacent PTMs are permissible and show high affinities, while some combinations weaken the acetyllysine interaction. Our novel X-ray structures demonstrate a unique binding mode of ATAD2B BRD compared to ATAD2. While the difference in ligand selectivity could suggest that ATAD2B may regulate different biological functions, our cellular assays, including western blot and functional genomic studies, indicate that ATAD2B is conserved in its ability to bind the chromatin. Finally, we discuss how known ‘onco’ mutations within the histone H4 tail region, occurring at or around sites of PTMs, can affect the ATAD2B BRD - acetyllysine coordination. Overall, our study highlights how crosstalk between various histone PTMs regulates the ‘reader’ domain of ATAD2B and affects its recruitment to the chromatin, thus providing new insights for developing therapeutic interventions for treating various diseases. Citation Format: Margaret Phillips, Kathleen Quinn, Cameron Montgomery, Samuel P. Boyson, Sunsik Chang, Jay C. Nix, Seth E. Frietze, Karen C. Glass. Insights into the histone code recognition by the ATAD2B bromodomain. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR002.

Cross‐talk between Histone Modifications Modulate Chromatin Reader Activity of the ATAD2B Bromodomain

The ATPase family, AAA domain‐containing protein 2 (ATAD2, or ANCCA) is an AAA nuclear co‐regulator protein containing two ATPase domains and a bromodomain. It’s closely related paralogue, ATAD2B (KIAA1240), also contains two ATPase domains and a bromodomain. The AAA ATPase domains are broadly associated with ATP driven molecular remodeling reactions while the bromodomains are evolutionarily conserved, chromatin ‘reader’ domains, known to regulate gene expression through recognition of histone post‐translational modifications (PTMs).Extensive studies on ATAD2 discuss its upregulation and correlation with poor prognosis in various cancers specially breast cancer and have successfully mapped the different histone interactions needed for targeting ATAD2 to the chromatin. Despite sharing high structural and sequence similarity with ATAD2, little is known about the unique role of ATAD2B. ATAD2B is expressed during neuronal differentiation and in tumor progression. However, limited information is available on how ATAD2B is involved or upregulated in various tumors specially in breast cancer. Our group has recently published a broad range of histone modifications recognized by the ATAD2B bromodomain. These modifications mostly include acetyllysine moieties on histone H4.Here, we present a more detailed structural and functional insight into how various histone post‐translational cross‐talk can modulate the chromatin ‘reader’ activity of ATAD2B bromodomain. Through ITC, solution NMR spectroscopy and X‐ray crystallography we demonstrate how combinations of PTMs can influence and regulate histone recognition by ATAD2B bromodomain. The studies also involve histone variants associated with breast cancer. These in‐vitro studies followed by in‐vivo analysis in breast cancer cells (MCF7) will further characterize the physiologically relevant, histone PTMs that are recognized by ATAD2B to direct it to the chromatin, thus playing a crucial role in activation of gene transcription.Through our study we highlight the importance of cross‐talk between various histone post‐translational modifications and how they may regulate the ATAD2B bromodomain activity. These combinations of PTMs can either promote or inhibit the affinity of ATAD2B to the chromatin and in turn regulate gene transcription. Understanding the molecular mechanisms driving these interactions will provide new insights for the development of therapeutic interventions for the treatment of various diseases.

Exploring ATAD2 bromodomain function in the dynamic epigenetic landscape

The ATPase family, AAA domain‐containing protein 2 (ATAD2) is a nuclear co‐regulator protein highly expressed in many unrelated cancers. ATAD2 overexpression is linked to poor outcomes in patients. ATAD2 contains a C‐terminal bromodomain that “reads” acetylated lysine post‐translational modifications (PTM) that occur on histone proteins present in the nucleosome core particle. The epigenetic landscape is dynamic and combinatorial, containing multiple types and numbers of modifications on the histone proteins at any given time. Yet, it is unknown how the presence of other modifications adjacent to acetylated lysine residues impact bromodomain protein recognition and function. We hypothesized that the presence of nearby post‐translational modifications including methylation and phosphorylation would modulate the ability of the ATAD2 bromodomain to recognize its acetylated lysine binding partners. Previously, we systemically screened for multiple PTM combinations recognized by the ATAD2 bromodomain using dCypher technology from EpiCypher. In our current study, we further characterized the interaction with these histone ligands using biophysical techniques including isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), and X‐ray crystallography. Our results indicate that the histone binding activity of the ATAD2 bromodomain is impacted by the presence of nearby PTMs. Our results provide new insights on how the bromodomain functions to target the ATAD2 protein to chromatin, and how this interaction may be fine‐tuned via dynamic changes in the epigenetic landscape.

Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain.

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.

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain.

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.

Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3.

Leydig cell steroidogenesis is a multistep process that takes place in the mitochondria and endoplasmic reticulum (ER). The physical association between these 2 organelles could facilitate both steroidogenesis substrate availability and mitochondrial product passage to steroidogenic enzymes in the ER, thus regulating the rate of steroid formation. Confocal microscopy, using antisera against organelle-specific antigens, and electron microscopy studies demonstrated that there is an increase in the number of mitochondria-ER contact sites in response to hormone treatment in MA-10 mouse tumor Leydig cells. Electron tomography and 3-dimensional reconstruction allowed for the visualization of mitochondria-associated membranes (MAMs). MAMs were isolated and found to contain the 67-kDa long isoform of the adenosine triphosphatase (ATPase) family, AAA domain-containing protein 3 (ATAD3). The 67-kDa ATAD3 is anchored in the inner mitochondrial membrane and is enriched in outer-inner mitochondrial membrane contact sites. ATAD3-depleted MA-10 cells showed reduced production of steroids in response to human choriogonadotropin but not to 22R-hydroxycholesterol treatment, indicating a role of ATAD3 in the delivery of the substrate cholesterol into the mitochondria. The N terminus of ATAD3 contains 50 amino acids that have been proposed to insert into the outer mitochondrial membrane and associated organelles such as the ER. Deletion of the ATAD3 N terminus resulted in the reduction of hormone-stimulated progesterone biosynthesis, suggesting a role of ATAD3 in mitochondria-ER contact site formation. Taken together, these results demonstrate that the hormone-induced, ATAD3-mediated, MAM formation participates in the optimal transfer of cholesterol from the ER into mitochondria for steroidogenesis.