Biology subjects

Markley, J. L.

Publications and source records attributed to Markley, J. L..

5 recordsLinked to original sources

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↗

bioRxiv2020

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.

biochemistry↗

bioRxiv2018

Robust nomenclature and software for enhanced reproducibility in molecular modeling of small molecules

Computational molecular dynamics, energy minimization, and modeling of molecular interactions are widely used in studies involving natural products, metabolites, and drugs. Manually directed computational steps commonly utilize an evolving collection of experimental and computational data, to which new data sources are added or modified as needed. Several software packages capable of incorporating sources of data are available, but the process remains error prone owing to the complexities of preparing and maintaining a consistent set of input files and the proper post-processing of derived data. We have devised a methodology and implemented it using an extensible software pipeline called RUNER (for Robust and Unique Nomenclature for Enhanced Reproducibility) that creates a robust and standardized computational process. The pipeline combines a web service and a graphical user interface (GUI) to enable seamless modifications and verified maintenance of atom force field parameters. The GUI provides an implementation for the widely used molecular modeling software package Xplor-NIH. We describe the RUNER software and demonstrate the rationale for the pipeline through examples of structural studies of small molecules and natural products. The software, pipeline, force field parameters, and file verification data for more than 4,100 compounds (including FDA-approved drugs and natural products) are freely accessible from [http://runer.nmrfam.wisc.edu].\n\nAuthor SummaryWe describe an automated and verifiable computational pipeline for calculating the force field parameters of small molecules. The pipeline integrates several software tools and guarantees reproducibility of the parameters by utilizing a standard nomenclature across multiple computational steps and by maintaining file verification identifiers. We demonstrate the application of this pipeline to (a) processing of more than 4,100 compounds in high-throughput mode, and (b) structural studies of natural products. The graphical user interface (GUI) associated with the pipeline facilitates the manually tedious steps of force field parameters adjustments and supports visualization of the process.

bioinformatics↗

bioRxiv2018

Inhibition of mitochondrial ferredoxin 1 (FDX1) prevents adaptation to proteotoxic stress

The mechanisms used by cancer cells to resist the severe disruption in protein homeostasis caused by proteasome inhibitors remain obscure. Here, we show this resistance correlates with a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Employing small molecule screens, we identified a striking overlap between compounds that preferentially impede the growth of proteasome inhibitor-resistant cancer cells and those that block the growth of high OXPHOS cells. Elesclomol potently exhibits both characteristics. Using genome-wide CRISPR/Cas9-based screening, in vitro validation and NMR spectroscopy we identify mitochondrial protein ferredoxin 1 (FDX1), a critical component of mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, as the primary target of elesclomol. In a mouse model of multiple myeloma, inhibition of FDX1 with elesclomol significantly attenuated the emergence of proteasome inhibitor-resistance and markedly prolonged survival. Our work reveals that the mitochondrial Fe-S cluster pathway is a targetable vulnerability in cancers that are resistant to increased proteotoxic burden.

cancer biology↗

bioRxiv2018

Structural insights into the recognition of mono- and di-acetyllysine by the ATAD2B bromodomain

Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 [A] resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin. HIGHLIGHTSO_LIThe ATAD2B bromodomain recognizes mono- and di-acetylated histone ligands. C_LIO_LIChemical shift perturbations outline the ATAD2B bromodomain acetyllysine binding pocket. C_LIO_LIAn ATAD2B bromodomain-inhibitor complex reveals important binding contacts. C_LIO_LIAn alternate splice variant in the ATAD2B bromodomain abolishes histone and inhibitor binding. C_LI

biochemistry↗