Cryo-EM of the Nucleosome Core Particle Bound to Ran-RCC1 Reveals a Dynamic Complex.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder-order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

Optimization of the synthesis of BET BD2 selective inhibitor XY153.

XY153 is a promising BET BD2 inhibitor with an IC50 value of 0.79 nM against BRD4 BD2. It shows 354-fold selectivity over BRD4-BD1 and 6-fold selectivity over other BET BD2 domains. However, the reported synthesis route of XY153 and its derivatives are extremely poor-yielding. After the synthesis of three key fragments, XY153 can only be obtained with a yield of 1.3 % in the original four-step reaction. In this study, we reported a three-step alternative route in the synthesis process of XY153. The reaction conditions for this route were thoroughly investigated and optimized, resulting in a significantly improved yield of 61.5 %. This efficient synthesis route establishes a robust chemical foundation for the rapid synthesis of XY153 derivatives as BET BD2 inhibitors in the near future.

Integrated virtual screening and MD simulation approaches toward discovering potential inhibitors for targeting BRPF1 bromodomain in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive and life-threatening cancers. Although multiple treatment options are available, the prognosis of HCC patients is poor due to metastasis and drug resistance. Hence, discovering novel targets is essential for better therapeutic development for HCC. In this study, we used the cancer genome atlas (TCGA) dataset to analyze the expression of bromodomain-containing proteins in HCC, as bromodomains are emerging attractive therapeutic targets. Our analysis identified BRPF1 as the most highly upregulated gene in HCC among the 43 bromodomain-containing genes. Upregulation of BRPF1 was significantly associated with poorer patient survival. Therefore, targeting BRPF1 may be an approach for HCC treatment. Previously, several potential inhibitors of BRPF1 bromodomain have been discovered. However, due to the limited clinical success of the current inhibitors, we aim to search for new inhibitors with high affinity and specificity for the BRPF1 bromodomain. In this study, we utilized high-throughput virtual screening methods to screen synthetic and natural compound databases against the BRPF1 bromodomain. In addition, we used machine learning-based QSAR modeling to predict the IC50 values of the selected BRPF1 bromodomain inhibitors. Extensive MD simulations were used to calculate the binding free energies of BRPF1 bromodomain and inhibitor complexes. Using this approach, we identified four lead scaffolds with a similar or better binding affinity towards the BRPF1 bromodomain than the previously reported inhibitors. Overall, this study discovered some promising compounds that have the potential to act as potent BRPF1 bromodomain inhibitors.

Leishmania PNUTS discriminates between PP1 catalytic subunits through an RVxF-ΦΦ-F motif and polymorphisms in the PP1 C-tail and catalytic domain.

Phosphoprotein phosphatase 1 (PP1) associates with specific regulatory subunits to achieve, among other functions, substrate selectivity. Among the eight PP1 isotypes in Leishmania, PP1-8e associates with the regulatory protein PNUTS along with the structural factors JBP3 and Wdr82 in the PJW/PP1 complex that modulates RNA polymerase II (pol II) phosphorylation and transcription termination. Little is known regarding interactions involved in PJW/PP1 complex formation, including how PP1-8e is the selective isotype associated with PNUTS. Here, we show that PNUTS uses an established RVxF-ΦΦ-F motif to bind the PP1 catalytic domain with similar interfacial interactions as mammalian PP1-PNUTS and noncanonical motifs. These atypical interactions involve residues within the PP1-8e catalytic domain and N and C terminus for isoform-specific regulator binding. This work advances our understanding of PP1 isoform selectivity and reveals key roles of PP1 residues in regulator binding. We also explore the role of PNUTS as a scaffold protein for the complex by identifying the C-terminal region involved in binding JBP3 and Wdr82 and impact of PNUTS on the stability of complex components and function in pol II transcription in vivo. Taken together, these studies provide a potential mechanism where multiple motifs within PNUTS are used combinatorially to tune binding affinity to PP1, and the C terminus for JBP3 and Wdr82 association, in the Leishmania PJW/PP1 complex. Overall, our data provide insights in the formation of the PJW/PP1 complex involved in regulating pol II transcription in divergent protozoans where little is understood.

The structural mechanism of dimeric DONSON in replicative helicase activation.

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

HTS discovery of PARP1-HPF1 complex inhibitors in cancer.

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.

Energy landscape quantifications of histone H3.3 recognition by chaperone DAXX reveal an uncoupled binding specificity and affinity.

Histone variant H3.3 differs from the canonical histone H3.1 by only five amino acids, yet its chaperone death domain-associated protein (DAXX) can specifically recognize H3.3 over H3.1, despite having a large DAXX-interacting surface on the H3.3-H4 heterodimer common to that on the H3.1-H4 complex. This observation gives rise to the question of, from the binding energy point view, how high binding specificity may be achieved with small differences of the overall binding energy for protein-protein interactions in general. Here we investigate the mechanism of coupling of binding specificity and affinity in protein-protein interactions using the DAXX-H3.3-H4 complex as a model. Using a multi-scale method, we found that the hydrophobic interactions between DAXX and the H3.3-specific region contributed to their initial binding process. And the structural flexibility of the interacting partners contributed to the binding affinity after their encounter. By quantifying the free energy landscape, we revealed that the interaction between the specific residues of H3.3 and DAXX decreased the encounter barrier height while the folding of H3.3-H4 and DAXX increased the depth of the free energy basin of the final binding state. The encounter barrier height, which is not coupled to the thermodynamic stability of the final binding state, had a marked effect on the initial binding rate of flexible histones and chaperones. Based on the energy landscape theory, we found that the intrinsic binding energy funnel of this uncoupled recognition process was affected by the structural flexibility and the flexibility modulated the degree of coupling between binding specificity and affinity. Our work offers a biophysical explanation of the specific recognition between the histones and their chaperones, and also extends the use of energy landscape theory for understanding molecular recognitions in general.

The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation.

Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a ß strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.

Catching proteins for degradation.

Aubiquitin-independent pathway targets nuclear proteins to the proteasome.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Recent advances in chromosome capture techniques unraveling 3D genome architecture in germ cells, health, and disease.

In eukaryotes, the genome does not emerge in a specific shape but rather as a hierarchial bundle within the nucleus. This multifaceted genome organization consists of multiresolution cellular structures, such as chromosome territories, compartments, and topologically associating domains, which are frequently defined by architecture, design proteins including CTCF and cohesin, and chromatin loops. This review briefly discusses the advances in understanding the basic rules of control, chromatin folding, and functional areas in early embryogenesis. With the use of chromosome capture techniques, the latest advancements in technologies for visualizing chromatin interactions come close to revealing 3D genome formation frameworks with incredible detail throughout all genomic levels, including at single-cell resolution. The possibility of detecting variations in chromatin architecture might open up new opportunities for disease diagnosis and prevention, infertility treatments, therapeutic approaches, desired exploration, and many other application scenarios.

Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.

Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27 kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

Electronegative clusters modulate folding status and RNA binding of unstructured RNA-binding proteins.

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA-binding affinity of SLBP to the sub-picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA-bound resembling state. We find that in the RNA-bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA-binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double-stranded RNA-binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA-binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface.

SARS-CoV-2 envelope protein attain Kac mediated dynamical interaction network to adopt 'histone mimic' at BRD4 interface.

Interface mimicry, achieved by recognition of host-pathogen interactions, is the basis by which pathogen proteins can hijack the host machinery. The envelope (E) protein of SARS-CoV-2 is reported to mimic the histones at the BRD4 surface via establishing the structural mimicry; however, the underlying mechanism of E protein mimicking the histones is still elusive. To explore the mimics at dynamic and structural residual network level an extensive docking, and MD simulations were carried out in a comparative manner between complexes of H3-, H4-, E-, and apo-BRD4. We identified that E peptide is able to attain an 'interaction network mimicry', as its acetylated lysine (Kac) achieves orientation and residual fingerprint similar to histones, including water-mediated interactions for both the Kac positions. We identified Y59 of E, playing an anchor role to escort lysine positioning inside the binding site. Furthermore, the binding site analysis confirms that E peptide needs a higher volume, similar to the H4-BRD4 where both the lysine's (Kac5 and Kac8) can accommodate nicely, however, the position of Kac8 is mimicked by two additional water molecules other than four water-mediated bridging's, strengthening the possibility that E peptide could hijack host BRD4 surface. These molecular insights seem pivotal for mechanistic understanding and BRD4-specific therapeutic intervention. KEY POINTSMolecular mimicry is reported in hijacking and then outcompeting the host counterparts so that pathogens can rewire their cellular function by overcoming the host defense mechanism.The molecular recognition process is the basis of molecular mimicry. The E peptide of SARS-CoV-2 is reported to mimic host histone at the BRD4 surface by utilizing its C-terminally placed acetylated lysine (Kac63) to mimic the N-terminally placed acetylated lysine Kac5GGKac8 histone (H4) by interaction network mimicry identified through microsecond molecular dynamics (MD) simulations and post-processing extensive analysis.There are two steps to mimic: firstly, tyrosine residues help E to anchor at the BRD4 surface to position Kac and increase the volume of the pocket. Secondary, after positioning of Kac, a common durable interaction network N140:Kac5; Kac5:W1; W1:Y97; W1:W2; W2:W3; W3:W4; W4:P82 is established between Kac5, with key residues P82, Y97, N140, and four water molecules through water mediate bridge. Furthermore, the second acetylated lysine Kac8 position and its interaction as polar contact with Kac5 were also mimicked by E peptide through interaction network P82:W5; W5:Kac63; W5:W6; W6:Kac63.The binding event at BRD4/BD1 seems an induced-fit mechanism as a bigger binding site volume was identified at H4-BRD4 on which E peptide attains its better stability than H3-BRD4.We identified the tyrosine residue Y59 of E that acts like an anchor on the BRD4 surface to position Kac inside the pocket and attain the interaction network by using aromatic residues of the BRD4 surface.Communicated by Ramaswamy H. Sarma.

Identification of nonhistone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

Abiotic peptides as carriers of information for the encoding of small-molecule library synthesis.

Encoding small-molecule information in DNA has been leveraged to accelerate the discovery of ligands for therapeutic targets such as proteins. However, oligonucleotide-based encoding is hampered by inherent limitations of information stability and density. In this study, we establish abiotic peptides for next-generation information storage and apply them for the encoding of diverse small-molecule synthesis. The chemical stability of the peptide-based tag allows the use of palladium-mediated reactions to efficiently synthesize peptide-encoded libraries (PELs) with broad chemical diversity and high purity. We demonstrate the successful de novo discovery of small-molecule protein ligands from PELs by affinity selection against carbonic anhydrase IX and the oncogenic protein targets BRD4(1) and MDM2. Collectively, this work establishes abiotic peptides as carriers of information for the encoding of small-molecule synthesis, leveraged herein for the discovery of protein ligands.

Investigation of novel ligand targeting bromodomain-containing protein 4 (BRD4) for cancer drug discovery: complete pharmacophore approach.

The Bromodomain (BRD4) and extra-terminal (BET) protein family are reversible; lysine-acetylated epigenetic readers identified as key important epigenetic regulators for protein recognition in posttranslational modifications for targeting cancer for its role in super-enhancers and transcription of oncogene expression in cancer and other forms of cancer and various diseases. Firstly, JQ-1a small potent BET inhibitors, targeting BET proteins were currently in clinical trials to ablate cancer. The identified compounds were taken from the library of preexisting therapeutically potent molecules. The objective of the present study is to identify the potential small molecule inhibitors against BRD4 through in-silico approach for the treatment of cancer. In present study, designed an in-silico screening of small molecules through ligand-based pharmacophore studies against bromodomain-containing protein 4 (BRD-4) protein and used for virtual screening through Database and their binding affinity and interaction of identified molecules were predicted through molecular docking, molecular dynamics simulations for 12 fixed time period, Molecular mechanics (MMGBSA) binding free energy calculations, ADME with drug-likeness properties including violations of lipinski's rule of 5, Jorgensens rule of 3 and other parameters were studied. The docking results indicate from the reported database screened molecules were validated with docking score -7.92 to -4.27Kcal/mol for BRD4-BD1 and the best model identified 21 hits. Among these two drugs were filtered and scrutinized for their ability based on binding modes and common interaction, MMGBSA of the highest affinity -54.53 Kcal/mol of BRD4-BD1 and ADME properties of selected molecules were predicted for its various parameters, dynamics studies evaluating its binding stability using Maestro software. In Conclusion, two BRD4 inhibitors were found to bind strongly in the similar binding sites as JQ-1, highlighting the role of BRD4-BD1. These compounds were identified as promising new options for regulating epigenetics and understanding the structural needs of BRD4 protein, further research in these areas could lead to the development of more effective and targeted cancer drugs.Communicated by Ramaswamy H. Sarma.