The ATAD2 bromodomain binds different acetylation marks on the histone H4 in similar fuzzy complexes.

Langini, Cassiano; Caflisch, Amedeo; Vitalis, Andreas

doi:10.1074/jbc.aac117.000630

Cited by 8 publications

(18 citation statements)

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“…In addition, the helical sections do not show any relevant motions at the backbone level with RMSF values around 0.5 Å for all but the C‐terminal helix. The highest fluctuations are observed for the ZA loop in agreement with previous simulation studies …”

Section: Resultssupporting

confidence: 92%

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

et al. 2018

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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). Sitedirected 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 ligandbinding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module. K E Y W O R D S acetyllysine, ATAD2, bromodomain inhibitor, chromatin reader domain, disulfide bridge, epigenetics, histone, post-translational modification

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Section: Resultssupporting

confidence: 92%

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

et al. 2018

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“…The H4K8ac (1-10) histone peptide binds with weakest affinity, similar to what was observed for other sub-family IV bromodomains, namely ATAD2B and BRPF1 [27] [42]. While the NMR CSPs confirm that the residues involved in the interaction of K5ac and K8ac are similar in ATAD2 BRD, it is the residues adjacent to the acetyllysine that control the histone peptide readout by the bromodomain and contribute to the resulting complex stability [3,33].…”

Section: Discussionsupporting

confidence: 71%

“…With majority of resonances assigned, NMR titration of mono-and di-acetylated histones into the ATAD2 BRD was valuable in defining a cluster of affected residues lining the binding pocket that are involved in the BRD-histone interaction. However, assignment of the ZA loop was difficult and limited due to high disorder in the "solventexposed" residues, resulting in signal broadening beyond detection [33].…”

Section: Discussionmentioning

confidence: 99%

“…A molecular dynamics study indicated that coordination of either the K5ac or the K12ac group of the H4K5acK12ac ligand within the ATAD2 BRD binding pocket is almost identical [2]. This also suggests that the ZA and BC loops remain partially disordered even after ligand binding, and this 'fuzzy' interaction may promote cooperativity in recognition of the first and second acetyllysines on the histone tail [33].…”

Section: Introductionmentioning

confidence: 99%

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Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

Evans

Phillips

Malone

et al. 2021

Preprint

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The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four alpha-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.

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“…There are, however, relevant precedents in the literature. Functional flexibility has been reported for acetyltransferases acting on histones [47], as well as bromo-domains binding such acetylation sites [48]. Flexibility and fluctuating conformations may shed light on how ASHH2 CW differentiates between methylation states while at the same time effectively searching the histones.…”

Section: Discussionmentioning

confidence: 99%

The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection

et al. 2020

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Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. b-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the g1, g3 and C-terminal coils, but also in the b1 and b2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome.

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The ATAD2 bromodomain binds different acetylation marks on the histone H4 in similar fuzzy complexes.

Cited by 8 publications

References 0 publications

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain

Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection

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