Inhibitors of the transactivation domain of androgen receptor as a therapy for prostate cancer.

The androgen receptor (AR) is a modular transcription factor which functions as a master regulator of gene expression. AR protein is composed of three functional domains; the ligand-binding domain (LBD); DNA-binding domain (DBD); and the intrinsically disordered N-terminal transactivation domain (TAD). AR is transactivated upon binding to the male sex hormone testosterone and other androgens. While the AR may tolerate loss of its LBD, the TAD contains activation function-1 (AF-1) that is essential for all AR transcriptional activity. AR is frequently over-expressed in most prostate cancer. Currently, androgen deprivation therapy (ADT) in the form of surgical or chemical castration remains the standard of care for patients with high risk localized disease, advanced and metastatic disease, and those patients that experience biochemical relapse following definitive primary treatment. Patients with recurrent disease that receive ADT will ultimately progress to lethal metastatic castration-resistant prostate cancer. In addition to ADT not providing a cure, it is associated with numerous adverse effects including cardiovascular disease, osteoporosis and sexual dysfunction. Recently there has been a renewed interest in investigating the possibility of using antiandrogens which competitively bind the AR-LBD without ADT for patients with hormone sensitive, non-metastatic prostate cancer. Here we describe a class of compounds termed AR transactivation domain inhibitors (ARTADI) and their mechanism of action. These compounds bind to the AR-TAD to inhibit AR transcriptional activity in the absence and presence of androgens. Thus these inhibitors may have utility in preventing prostate cancer growth in the non-castrate setting.

Dichotomous transactivation domains contribute to growth inhibitory and promotion functions of TAp73.

The transcription factor p73, a member of the p53 tumor-suppressor family, regulates cell death and also supports tumorigenesis, although the mechanistic basis for the dichotomous functions is poorly understood. We report here the identification of an alternate transactivation domain (TAD) located at the extreme carboxyl (C) terminus of TAp73ß, a commonly expressed p73 isoform. Mutational disruption of this TAD significantly reduced TAp73ß's transactivation activity, to a level observed when the amino (N)-TAD that is similar to p53's TAD, is mutated. Mutation of both TADs almost completely abolished TAp73ß's transactivation activity. Expression profiling highlighted a unique set of targets involved in extracellular matrix-receptor interaction and focal adhesion regulated by the C-TAD, resulting in FAK phosphorylation, distinct from the N-TAD targets that are common to p53 and are involved in growth inhibition. Interestingly, the C-TAD targets are also regulated by the oncogenic, amino-terminal-deficient DNp73ß isoform. Consistently, mutation of C-TAD reduces cellular migration and proliferation. Mechanistically, selective binding of TAp73ß to DNAJA1 is required for the transactivation of C-TAD target genes, and silencing DNAJA1 expression abrogated all C-TAD-mediated effects. Taken together, our results provide a mechanistic basis for the dichotomous functions of TAp73 in the regulation of cellular growth through its distinct TADs.

Assessment of machine-learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains.

The human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces, named H1 and H2, which are found on opposite sides of its ACID domain. Here, we use and compare deep learning methods to characterize human MED25-TAD interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high-reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of MED25 interface predictions with the viral transcriptional activators Lana-1 and IE62. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for the Arabidopsis MED25 ACID domain.

Commonly asked questions about transcriptional activation domains.

Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing structurally related DNA sequences; they are structured, conserved, and predictable from protein sequences. Activation domains recruit chromatin modifiers, coactivator complexes, or basal transcriptional machinery via structurally diverse protein-protein interactions. Activation domains and DNA-binding domains have been called independent, modular units, but there are many departures from modularity, including interactions between these regions and overlap in function. Compared to DNA-binding domains, activation domains are poorly understood because they are poorly conserved, intrinsically disordered, and difficult to predict from protein sequences. This review, organized around commonly asked questions, describes recent progress that the field has made in understanding the sequence features that control activation domains and predicting them from sequence.

[Understanding the Underlying Mechanism of Xenobiotic-Sensing Nuclear Receptor Activation].

The nuclear receptor superfamily comprises 48 members in humans. In various organs, nuclear receptors regulate a variety of physiological functions through transcription of target genes. They are associated with the development and progression of endocrine and metabolic disorders, as well as with cancer development. Therefore, agonists and antagonists targeting nuclear receptors are currently being developed as therapeutic drugs for these diseases. Nuclear receptors can be activated through ligand binding or phosphorylation, which is mediated by various cellular signaling pathways. Activation of a nuclear receptor necessitates significant structural modifications in each of its domains. My research has been focused on unraveling the intricate mechanisms underlying the activation of nuclear receptors using constitutive androstane receptor (CAR) and pregnane X receptor (PXR) as model nuclear receptor proteins. CAR and PXR are highly expressed in the liver and are activated by a wide range of xenobiotics. Given their crucial roles in the metabolism and disposition of xenobiotics, as well as their potential in mediating drug-drug interactions, it is imperative to extensively study the mechanisms of xenobiotic-induced activation of these receptors. Such studies are essential for advancements in drug development, as well as for ensuring food and chemical safety. In this review, I elucidate the molecular basis underlying the activation of xenobiotic-responsive nuclear receptors.

NF-YA isoforms with alternative splicing of exon-5 in Aves.

NF-YA, the regulatory subunit of the trimeric CCAAT-binding transcription factor NF-Y, is present in vertebrates in two major alternative spliced isoforms: NF-YAl and NF-YAs, differing for the presence of exon-3. NF-YAx, a third isoform without exon-3/-5, was reported only in human neuronal cells and tumors. These events affect the Trans-Activation Domain. We provide here evidence for the expression of NF-YAx and for the existence of a new isoform, NF-YAg, skipping only exon-5. These isoforms are abundant in Aves, but not in reptiles, and are the prevalent transcripts in the initial phases of embryo development in chicken. Finally, we analyzed NF-YAg and NF-YAx amino acid sequence using AlphaFold: absence of exon-5 denotes a global reduction of ß-stranded elements, while removal of the disordered exon-3 sequence has limited effects on TAD architecture. These data identify an expanded program of NF-YA isoforms within the TAD in Aves, implying a role during early development.

Clusters of acidic and hydrophobic residues can predict acidic transcriptional activation domains from protein sequence.

Transcription factors activate gene expression in development, homeostasis, and stress with DNA binding domains and activation domains. Although there exist excellent computational models for predicting DNA binding domains from protein sequence, models for predicting activation domains from protein sequence have lagged, particularly in metazoans. We recently developed a simple and accurate predictor of acidic activation domains on human transcription factors. Here, we show how the accuracy of this human predictor arises from the clustering of aromatic, leucine, and acidic residues, which together are necessary for acidic activation domain function. When we combine our predictor with the predictions of convolutional neural network (CNN) models trained in yeast, the intersection is more accurate than individual models, emphasizing that each approach carries orthogonal information. We synthesize these findings into a new set of activation domain predictions on human transcription factors.

Prostaglandins as Candidate Ligands for a Per-ARNT-Sim (PAS) Domain of Steroid Receptor Coactivator 1 (SRC1).

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor.

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

PTIP Deficiency in B Lymphocytes Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

BACKGROUND: Immune dysregulation contributes to the development of ulcerative colitis (UC). The research on the inflammatory response of UC is mainly focused on T cells, with less understanding of the role of B cells. Pax transactivation domain-interacting protein (PTIP) is essential for the development of B cell subpopulations and humoral immunity. The purpose of this study was to elucidate the role of PTIP in B cells of mice with dextran sodium sulfate (DSS)-induced colitis. METHODS: The B-cell-specific PTIP knockout (PTIP-/-) mice were established by crossbreeding cluster of differentiation (CD)19cre/cre mice with PTIPflox/flox mice. The UC mice were induced by drinking water supplemented with 3.8% Dextran Sulfate Sodium (DSS) (PTIP-/- + DSS). The histological analysis was performed using hematoxylin and eosin staining. The immune cells were isolated using a fluorescence-activated cell sorter. The serum antibodies (immunoglobulin M (IgM) or immunoglobulin G (IgG)) and tumor necrosis factor (TNF)-α were determined by Enzyme linked immunosorbent assay (ELISA). RESULTS: Interestingly, our findings demonstrate that PTIP deficiency in B cells significantly ameliorates UC. In contrast to PTIP-/- + DSS, the wild type (WT) + DSS group showed a more robust increase in disease activity index (DAI) scores (p < 0.05), a substantially shortened colon (p < 0.001) and a decrease of mucous-producing goblet cells and the complete destruction of crypts. Moreover, PTIP-deficient mice manifested markedly altered neutrophil and T-cell distribution in UC (p < 0.05). Although anti-commensal IgG exacerbates UC, we demonstrated, for the first time, that serum natural IgG does not aggravate the pathology of UC. Furthermore, PTIP regulates UC by controlling B-2 cells independently from T cells. CONCLUSIONS: Transplantation of splenic B-2 cells from PTIP-deficient mice protected recipient NOD/ShiltJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt (NCG) mice from severe UC.

ß-Catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription.

The transcriptional co-regulator ß-catenin is a critical member of the canonical Wnt signaling pathway, which plays an important role in regulating cell fate. Deregulation of the Wnt/ß-catenin pathway is characteristic in the development of major types of cancer, where accumulation of ß-catenin promotes cancer cell proliferation and renewal. ß-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of ß-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of ß-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal ß-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to ß-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results outline a mechanism of Wnt/ß-catenin gene regulation that underlies cell development and provides a framework to develop methods to block ß-catenin dependent signaling.

FOXO transcription factors differ in their dynamics and intra/intermolecular interactions.

Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would modulate their activities by interacting mutually.

Multifaceted regulation and functions of 53BP1 in NHEJ­mediated DSB repair (Review).

The repair of DNA double­strand breaks (DSBs) is crucial for the preservation of genomic integrity and the maintenance of cellular homeostasis. Non­homologous DNA end joining (NHEJ) is the predominant repair mechanism for any type of DNA DSB during the majority of the cell cycle. NHEJ defects regulate tumor sensitivity to ionizing radiation and anti­neoplastic agents, resulting in immunodeficiencies and developmental abnormalities in malignant cells. p53­binding protein 1 (53BP1) is a key mediator involved in DSB repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. 53BP1 promotes DSB repair via NHEJ and antagonizes DNA end overhang resection. At present, novel lines of evidence have revealed the molecular mechanisms underlying the recruitment of 53BP1 and DNA break­responsive effectors to DSB sites, and the promotion of NHEJ­mediated DSB repair via 53BP1, while preventing homologous recombination. In the present review article, recent advances made in the elucidation of the structural and functional characteristics of 53BP1, the mechanisms of 53BP1 recruitment and interaction with the reshaping of the chromatin architecture around DSB sites, the post­transcriptional modifications of 53BP1, and the up­ and downstream pathways of 53BP1 are discussed. The present review article also focuses on the application perspectives, current challenges and future directions of 53BP1 research.

Phylogeny of NF-YA trans-activation splicing isoforms in vertebrate evolution.

NF-Y is a trimeric pioneer Transcription Factor (TF) whose target sequence -the CCAAT box- is present in ~25% of mammalian promoters. We reconstruct the phylogenetic history of the regulatory NF-YA subunit in vertebrates. We find that in addition to the remarkable conservation of the subunits-interaction and DNA-binding parts, the Transcriptional Activation Domain (TAD) is also conserved (>90% identity among bony vertebrates). We infer the phylogeny of the alternatively spliced exon-3 and partial splicing events of exon-7 -7N and 7C- revealing independent clade-specific losses of these regions. These isoforms shape the TAD. Absence of exon-3 in basal deuterostomes, cartilaginous fishes and hagfish, but not in lampreys, suggests that the "short" isoform is primordial, with emergence of exon-3 in chordates. Exon 7N was present in the vertebrate common ancestor, while 7C is a molecular innovation of teleost fishes. RNA-seq analysis in several species confirms expression of all these isoforms. We identify 3 blocks of amino acids in the TAD shared across deuterostomes, yet structural predictions and sequence analyses suggest an evolutionary drive for maintenance of an Intrinsically Disordered Region -IDR- within the TAD. Overall, these data help reconstruct the logic for alternative splicing of this essential eukaryotic TF.

MUL1-RING recruits the substrate, p53-TAD as a complex with UBE2D2-UB conjugate.

The RING domain of MUL1 (RINGMUL1 ) alone mediates ubiquitylation of the p53-transactivation domain (TADp53 ). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TADp53 by RINGMUL1 , we determined the complex structure of RINGMUL1 :UBE2D2 and studied the interaction between RINGMUL1 and TADp53 in the presence of UBE2D2-UB thioester (UBE2D2~UB) mimetics. The RINGMUL1 -binding induced the closed conformation of UBE2D2S22R/C85S -UBK48R oxyester (UBE2D2RS -UBR OE ), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A-mutation of UBE2D2. Interestingly, UBE2D2S22R/N77A/C85S -UBK48R oxyester (UBE2D2RAS -UBR OE ) already formed a closed conformation in the absence of RINGMUL1 . Although TADp53 exhibited weak binding for RINGMUL1 or UBE2D2 alone, its binding affinity was enhanced and even further for RINGMUL1 :UBE2D2 and RINGMUL1 :UBE2D2RAS -UBR OE , respectively. The recognition of TADp53 by RINGMUL1 as a complex with UBE2D2~UB is related to the multivalency of the binding events and underlies the ability of RINGMUL1 to ubiquitylate the intrinsically disordered protein, TADp53 .

Characterization of the transactivation and nuclear localization functions of Pichia pastoris zinc finger transcription factor Mxr1p.

The zinc finger transcription factor Mxr1p regulates the transcription of genes involved in methanol, acetate, and amino acid metabolism of the industrial yeast Pichia pastoris (a.k.a. Komagataella phaffii) by binding to Mxr1p response elements in their promoters. Here, we demonstrate that Mxr1p is a key regulator of ethanol metabolism as well. Using transcriptomic analysis, we identified target genes of Mxr1p that mediate ethanol metabolism, including ALD6-1 encoding an aldehyde dehydrogenase. ALD6-1 is essential for ethanol metabolism, and the ALD6-1 promoter harbors three Mxr1p response elements to which Mxr1p binds in vitro and activates transcription in vivo. We show that a nine-amino acid transactivation domain located between amino acids 365 and 373 of Mxr1p is essential for the transactivation of ALD6-1 to facilitate ethanol metabolism. Mxr1N250, containing the N-terminal 250 amino acids of Mxr1p, localized to the nucleus of cells metabolizing ethanol dependent on basic amino acid residues present between amino acids 75 and 85. While the N-terminal 400 amino acids of Mxr1p are sufficient for the activation of target genes essential for ethanol metabolism, the region between amino acids 401 and 1155 was also required for the regulation of genes essential for methanol metabolism. Finally, we identified several novel genes whose expression is differentially regulated by Mxr1p during methanol metabolism by DNA microarray. This study demonstrates that Mxr1p is a key regulator of ethanol metabolism and provides new insights into the mechanism by which Mxr1p functions as a global regulator of multiple metabolic pathways of P. pastoris.

Helix 12 stabilization contributes to basal transcriptional activity of PXR.

Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening.

The genomic regulatory elements for estrogen receptor alpha transactivation-function-1 regulated genes.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription regulator, containing two transactivation functional domains, AF-1 and AF-2. The selective estrogen receptor modulators (SERMs), including 4-hydroxytamoxifen (4OHT), activate AF-1 preferentially rather than AF-2. However, it is unclear whether this specific function is related to the tissue-selective functionality of SERMs. Moreover, there is no information determining AF-1-dependent estrogenic-genes existing in tissues. We sought to identify AF-1-dependent estrogenic-genes using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an AF-2 disrupted estradiol (E2)-insensitive mutant ERα, but AF-1-dependent transcription can be activated by the estrogen-antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP-Seq analysis identified Klk1b21 as an ICI-inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell-based reporter assay. The 5'-flanking 0.4k bp region of Klk1b21 gene responded as an ERα AF-1-dependent estrogen-responsive promoter. The 150 bp minimum ERα binding element (EBE) consists of three direct repeats. These three half-site sequences were essential for the ERα-dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine-kinase promoter but could be restored by fusion with the 100 bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF-1-dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF-1 dominant estrogenic-genes exist in estrogen-responsive organs. Such findings will aid in understanding the mechanism of ERα-dependent tissue-selective activity of SERMs.

Dissecting the transactivation domain (tAD) of the transcription factor c-Myb to assess recent models of tAD function.

Transcription factors use a DNA-binding domain to localize their action and a transactivation domain (tAD) to stimulate activation of the associated gene. Recent work has renewed interest in how tADs activate genes, which remains poorly understood. Key features in the new models are exposure of short linear motifs (SLMs) and liquid-liquid phase separation (LLPS). Inspired by the new models for tAD function, we decided to revisit the tAD of the haematopoietic transcription factor c-Myb by performing a mutational analysis to see how these new models fit and potentially explain the tAD behaviour of this master regulator. We know that c-Myb has an acidic tAD, which contains a well-characterized SLM in the form of a LxxLL motif. By testing 12 alanine-scanning mutants and three mutants with major reorganization of its tAD in two mammalian reporter systems, we found a pattern of effects very close to what would be expected from the SLM-exposure model, with strong effects exerted by both acidic replacements and SLM mutation. When the same mutants were tested in a yeast system, the pattern of effects was dramatically different, with the SLM mutation exerting no effect, and tAD behaviour was much less affected by small alterations, as would be expected from a LLPS model. These observations are discussed in the light of the two new tAD models, and a two-step hypothesis for transactivation, combining both models, is proposed.

Multivalent and Bidirectional Binding of Transcriptional Transactivation Domains to the MED25 Coactivator.

The human mediator subunit MED25 acts as a coactivator that binds the transcriptional activation domains (TADs) present in various cellular and viral gene-specific transcription factors. Previous studies, including on NMR measurements and site-directed mutagenesis, have only yielded low-resolution models that are difficult to refine further by experimental means. Here, we apply computational molecular dynamics simulations to study the interactions of two different TADs from the human transcription factor ETV5 (ERM) and herpes virus VP16-H1 with MED25. Like other well-studied coactivator-TAD complexes, the interactions of these intrinsically disordered domains with the coactivator surface are temporary and highly dynamic ('fuzzy'). Due to the fact that the MED25 TAD-binding region is organized as an elongated cleft, we specifically asked whether these TADs are capable of binding in either orientation and how this could be achieved structurally and energetically. The binding of both the ETV5 and VP16-TADs in either orientation appears to be possible but occurs in a conformationally distinct manner and utilizes different sets of hydrophobic residues present in the TADs to drive the interactions. We propose that MED25 and at least a subset of human TADs specifically evolved a redundant set of molecular interaction patterns to allow binding to particular coactivators without major prior spatial constraints.