Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
James M. Stafford
Abstract
The ATPase family, AAA+ domain-containing proteins, ATAD2 and ATAD2B, are the only human proteins known to contain a bromodomain fused to a AAA+ ATPase (ATPases Associated with diverse cellular Activities) domain. AAA+ ATPases typically function as molecular motors, while bromodomains are chromatin readers that recognize acetylated lysine residues on histone proteins. ATAD2 and ATAD2B likely contribute to the epigenetic regulation cellular processes; however, their molecular functions, and the roles of their conserved domains are unknown. This information is critical as dysregulation of ATAD2 and ATAD2B are linked to multiple types of cancer, and the activity of ATAD2B has also been implicated in neurological disorders and respiratory disease.In this work, we purified the bromodomains of ATAD2 and ATAD2B and tested their binding activity towards histone proteins containing multiple post-translational modifications (PTMs) such as acetylation, methylation, and phosphorylation. High- throughput peptide arrays were used to detect binding interactions between the bromodomains and histone ligands containing single or combinatorial modifications. We discovered that the bromodomains of ATAD2 and ATAD2B preferentially recognize acetylated histone H4, but ATAD2B appears to have a broader substrate specificity than ATAD2. Using a combination of biochemical and structural biology techniques including isothermal titration calorimetry, nuclear magnetic resonance, and X-ray crystallography, we characterized specific determinants driving histone recognition by the ATAD2 and ATAD2B bromodomains. We found that the ATAD2B bromodomain can accommodate multiple combinations of modifications within its binding pocket, and is unaffected by the presence of nearby PTMs to its acetyllysine binding partner. On the other hand, ATAD2 is selective for combinations of acetyllysine modifications and is inhibited by the presence of hydrophobic methylation or bulky phosphorylation PTMs. To investigate how the acetylated histone binding activity of the bromodomain functions in the context of the entire protein, which also contains two AAA+ ATPase domains, we developed a method to express and purify the nearly full-length ATAD2B protein. Although this construct is missing the unstructured N-terminal region, DN-ATAD2B forms a functional enzymatic complex in vitro, capable of hydrolyzing ATP. Utilizing cryo-electron microscopy (cryo- EM), we determined the structure of ATAD2B to a resolution of 3.0 Å, which revealed that ATAD2B assembles into a hexamer where the subunits are oriented in a shallow spiral that is stabilized through “knob” and “hole” interactions between adjacent monomers. In summary, our findings provide new insights into the function of the conserved domains within the ATAD2 and ATAD2B AAA+ ATPases. Specifically, the bromodomains coordinate different subsets of acetylated histones, while the AAA+ domains contribute to the formation of a hexameric complexes and enzymatic ATPase activity. In addition, our new knowledge about the three-dimensional organization of the functional domains within the ATAD2B complex will provide the basis for future experiments examining the chromatin regulatory functions of ATAD2 and ATAD2B. In the context of human disease, the bromodomains of ATAD2 and ATAD2B are attractive drug targets, and our structural studies provide critical information for the development of new therapeutics.
Language
en
Number of Pages
310 p.
Recommended Citation
Malone, Kiera Lenore, "Functional And Structural Characterization Of Atad2 And Atad2b Conserved Domains: Insights Into Epigenetic Histone Interaction And Atpase Function" (2025). Graduate College Dissertations and Theses. 2064.
https://scholarworks.uvm.edu/graddis/2064