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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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors-the magnesium ion cofactor and the protein substrate-on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery. [ABSTRACT FROM AUTHOR]

Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin- biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and sitespecific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies. [ABSTRACT FROM AUTHOR]

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 53-57 β-strand dissociation from TEAD4 and contribute to the lower affinity of YAP for TEAD4. Identifying a clear mechanism leading to the difference in YAP's β-strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity., (© 2022 The Protein Society.)

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., (© 2021 The Protein Society.)

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)., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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., (© 2019 The Protein Society.)