Abstract
ATPase family AAA+ domain-containing protein 2B (ATAD2B) is a poorly characterized member of the ATAD2-like protein family, which contains a unique combination of tandem AAA+ ATPase domains with a C-terminal bromodomain. In humans, ATAD2B is dysregulated in several disease states including cancer and respiratory disorders, yet despite its promise as a therapeutic target, little is known about its molecular function. Here, we report the first high-resolution cryo-EM structure of human ATAD2B at 3.0 Å, revealing a two-tiered hexameric assembly with a shallow spiral staircase architecture. Structural analysis uncovers conserved AAA+ ATPase features, including nucleotide coordination at inter-subunit interfaces, inter-subunit signaling (ISS) gate loops, and pore loops that engage a substrate within the central channel. Biochemical assays demonstrate that ATAD2B is an active enzyme with an ATP hydrolysis rate of 0.34 ATP/hexamer/sec. Furthermore, the integrity of the hexameric complex is stabilized through unique knob-hole interactions, a linker arm that extends between the AAA2 and bromodomain, and an N-terminal linker domain (LD). These findings establish ATAD2B as a functional AAA+ ATPase and provide mechanistic insight into its enzymatic activities, laying the foundation for understanding its role in chromatin-associated processes.