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The yes-associated protein (YAP) regulates the transcriptional activity of the TEAD transcription factors that are key in the control of organ morphogenesis. YAP interacts with TEAD via three secondary structure elements: a β-strand, an α-helix, and an Ω-loop. Earlier results have shown that the β-strand has only a marginal contribution in the YAP:TEAD interaction, but we show here that it significantly enhances the affinity of YAP for the Drosophila homolog of TEAD, scalloped (Sd). Nuclear magnetic resonance shows that the β-strand adopts a more rigid conformation once bound to Sd; pre-steady state kinetic measurements show that the YAP:Sd complex is more stable. Although the crystal structures of the YAP:TEAD and YAP:Sd complexes reveal no differences at the binding interface that could explain these results. Molecular Dynamics simulations are in line with our experimental findings regarding β-strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that β-sheet fluctuations play a relevant role in YAP

The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes‐associated protein), TAZ (transcriptional co‐activator with PDZ‐binding motif) and VGLL1‐4 (Vestigial‐like members 1–4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω‐loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full‐length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.

The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full‐length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high‐affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high‐affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.

The Hippo signaling pathway, which plays a central role in the control of organ size in animals, is well conserved in metazoans. The most downstream elements of this pathway are the TEAD transcription factors that are regulated by their association with the transcriptional coactivator YAP. Therefore, the creation of the binding interface that ensures the formation of the YAP:TEAD complex is a critical molecular recognition event essential for the development/survival of many living organisms. In this report, using the available structural information on the YAP:TEAD complex, we study the TEAD‐binding domain of YAP from different animal species. This analysis of more than 400 amino acid sequences reveals that the residues from YAP involved in the formation of the two main contact regions with TEAD are very well conserved. Therefore, the binding interface between YAP and TEAD, as found in humans, probably appeared at an early evolutionary stage in metazoans. We find that, in contrast to most other animal species, several Actinopterygii species possess YAP variants with a different TEAD‐binding domain. However, these variants bind to TEAD with a similar affinity. Our studies show that the protein identified as a YAP homolog in Caenorhabditis elegans does not contain the TEAD‐binding domain found in YAP of other metazoans. Finally, we do not identify in non‐metazoan species, amino acid sequences containing both a TEAD‐binding domain, as in metazoan YAP, and WW domain(s).

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer., Lineage-restricted transcription factor PAX8 is oncogenic in ovarian cancer cells. Here the authors show that PAX8 interacts and recruits a splice variant of the MECOM locus PRDM3 to control the gene expression module involved in adhesion and extracellular matrix, and consequently promotes ovarian tumorigenesis.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

The Hippo pathway is deregulated in various cancers, and the discovery of molecules that modulate this pathway may open new therapeutic avenues in oncology. TEA/ATTS domain (TEAD) transcription factors are the most distal elements of the Hippo pathway and their transcriptional activity is regulated by the Yes-associated protein (YAP). Amongst the various possibilities for targeting this pathway, inhibition of the YAP:TEAD interaction is an attractive strategy. It has been shown recently that TEAD proteins are covalently linked via a conserved cysteine to a fatty acid molecule (palmitate) that binds to a deep hydrophobic cavity present in these proteins. This acylation of TEAD seems to be required for efficient binding to YAP, and understanding how it modulates the YAP:TEAD interaction may provide useful information on the regulation of TEAD function. In this report we have studied the effect of TEAD4 acylation on its interaction with YAP and the other co-activator transcriptional co-activator with PDZ-binding motif (TAZ). We show in our biochemical and cellular assays that YAP and TAZ bind in a similar manner to acylated and non-acylated TEAD4. This indicates that TEAD4 acylation is not a prerequisite for its interaction with YAP or TAZ. However, we observed that TEAD4 acylation significantly enhances its stability, suggesting that it may help this transcription factor to acquire and/or maintain its active conformation.

The TEAD (TEA/ATTS domain) transcription factors are regulated by various coactivator proteins. A β-strand:loop:α-helix motif is present at the TEAD binding site of all the coactivators crystallized so far. These motifs interact with the same area of TEAD, suggesting that the coactivators compete with each other in vivo to gain access to TEAD. The α-helix, which shows marked interactions with TEAD, is the key element of the β-strand:loop:α-helix motif. A very large difference in potency (>40 fold) has been measured between the isolated mouse VGLL1 (vestigial-like 1, mVGLL1) α-helix and its human YAP (Yes-associated protein, hYAP) equivalent. Elucidating the mechanisms at the origin of this difference should help in better understanding how these coactivators interact with TEAD. In this report, we show that the β-strand:loop:α-helix motif of hYAP and mVGLL1 are optimized in a very different manner suggesting a convergent evolution of these coactivators for binding to the TEAD transcription factors.

Four TEAD transcription factors (TEAD1-4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson's chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol-1 . We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H-bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD 1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex. DATABASE: The crystallographic data have been deposited at the RSCB Protein Data Bank (PDB, www.pdb.org) with the access codes: 6HIL (wtYAP :Tyr421HisTEAD1 ), 6HIK (wtYAP :Tyr429HisTEAD4 ).

Many interactions between proteins are mediated by intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) do not adopt a stable three‐dimensional structure in their unbound form, but they become more structured upon binding to their partners. In this communication, we study how a bound IDR adapts to mutations, preventing the formation of hydrogen bonds at the binding interface that needs a precise positioning of the interacting residues to be formed. We use as a model the YAP:TEAD interface, where one YAP (IDP) and two TEAD residues form hydrogen bonds via their side chain. Our study shows that the conformational flexibility of bound YAP and the reorganization of water molecules at the interface help to reduce the energetic constraints created by the loss of H‐bonds at the interface. The residual flexibility/dynamic of bound IDRs and water might, therefore, be a key for the adaptation of IDPs to different interface landscapes and to mutations occurring at binding interfaces.

The Hippo pathway is deregulated in various cancers, and the discovery of molecules that modulate this pathway may open new therapeutic avenues in oncology. TEA/ATTS domain (TEAD) transcription factors are the most distal elements of the Hippo pathway and their transcriptional activity is regulated by the Yes‐associated protein (YAP). Amongst the various possibilities for targeting this pathway, inhibition of the YAP:TEAD interaction is an attractive strategy. It has been shown recently that TEAD proteins are covalently linked via a conserved cysteine to a fatty acid molecule (palmitate) that binds to a deep hydrophobic cavity present in these proteins. This acylation of TEAD seems to be required for efficient binding to YAP, and understanding how it modulates the YAP:TEAD interaction may provide useful information on the regulation of TEAD function. In this report we have studied the effect of TEAD4 acylation on its interaction with YAP and the other co‐activator transcriptional co‐activator with PDZ‐binding motif (TAZ). We show in our biochemical and cellular assays that YAP and TAZ bind in a similar manner to acylated and non‐acylated TEAD4. This indicates that TEAD4 acylation is not a prerequisite for its interaction with YAP or TAZ. However, we observed that TEAD4 acylation significantly enhances its stability, suggesting that it may help this transcription factor to acquire and/or maintain its active conformation.

TEAD (TEA/ATTS domain) transcription factors are the most distal effectors of the Hippo pathway. YAP (Yes-associated protein) is a coactivator protein which, upon binding to TEAD proteins, stimulates their transcriptional activity. Since the Hippo pathway is deregulated in various cancers, designing inhibitors of the YAP:TEAD interaction is an attractive therapeutic strategy for oncology. Understanding the molecular events that take place at the YAP:TEAD interface is therefore important not only to devise drug discovery approaches, but also to gain knowledge on TEAD regulation. In this report, combining single site-directed mutagenesis and double mutant analyses, we conduct a detailed analysis on the role of several residues located at the YAP:TEAD interface. Our results provide quantitative understanding of the interactions taking place at the YAP:TEAD interface and give insights into the formation of the YAP:TEAD complex and more particularly on the interaction between TEAD and the Ω-loop found in YAP. DOI: http://dx.doi.org/10.7554/eLife.25068.001

We describe a cloning and expression system which is based on the Escherichia coli T7 expression system and Gateway recombination technology. We have produced numerous destination vectors with selected fusion tags and an additional set of entry vectors containing the gene of interest and optional labeling tags. This powerful system enables us to transfer a cDNA to several expression vectors in parallel and combine them with various labeling tags. To remove the attached amino terminal tags along with the unwanted attB1 site, we inserted PreScission protease cleavage sites. In contrast to the commercially available destination vectors, our plasmids provide kanamycin resistance, which can be an advantage when expressing toxic proteins in E. coli. Some small-scale protein expression experiments are shown to demonstrate the usefulness of these novel Gateway vectors. In summary, this system has some benefits over the widely used and commercially available Gateway standard system, and it enables many different combinations for expression constructs from a single gene of interest.