Biology subjects

Malone, K. L.

Publications and source records attributed to Malone, K. L..

4 recordsLinked to original sources

bioRxiv2026

Cryo-EM Structure of Human ATAD2B Reveals a Hexameric Organization Contributes to ATPase Activity and Substrate Coordination

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 [A], 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.

biochemistry↗

bioRxiv2025

Breaking Barriers: Transitioning from X-ray Crystallography to Cryo-EM for Structural Studies of ATAD2B

Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution of large macromolecular complexes without the need for crystallization. Here, we describe our laboratorys transition from X-ray crystallography to single-particle cryo-EM to investigate the ATPase family AAA+ domain-containing protein 2B (ATAD2B), a chromatin regulator implicated in epigenetic signaling. We outline the challenges encountered during protein expression, purification, and sample preparation, including co-purification of the chaperonin GroEL, and strategies employed to overcome these obstacles. Our workflow highlights critical steps in sample optimization, grid vitrification, and data processing using CryoSPARC, cisTEM, and Topaz, as well as computational requirements for high-resolution reconstructions. We also discuss model building, refinement, and validation approaches, emphasizing best practices for new cryo-EM users. This work provides practical insights for structural biologists adopting cryo-EM, particularly for large, flexible protein complexes, and underscores the importance of integrated approaches combining biochemical, computational, and imaging strategies.

biochemistry↗

bioRxiv2022

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to up-regulate and down-regulate certain genes required for invasion and replication. The P. falciparum genome encodes six bromodomaincontaining proteins, of which Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and the 8 human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

biophysics↗

bioRxiv2021

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 -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.

biochemistry↗