RESUMO
The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.
Assuntos
Proteínas de Ciclo Celular/química , Complexos Multiproteicos/química , Fatores de Transcrição/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
The Hippo signaling pathway, which controls organ size in animals, is altered in various human cancers. The TEAD transcription factors, the most downstream elements in this pathway, are regulated by different cofactors, such as the Vgll (vestigial-like) proteins. Having studied the interaction between Vgll1-derived peptides and human TEAD4, we show that, although it lacks a key secondary structure element required for tight binding by two other TEAD cofactors (YAP and TAZ), Vgll1-derived peptides bind to TEAD with nanomolar affinity. We identify a ß-strand:loop:α-helix motif as the minimal Vgll binding site. Finally, we reveal an unexpected difference between mouse and human Vgll1-derived peptides.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Musculares/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.
Assuntos
DNA Helicases , Tiadiazóis , Ureia , DNA Helicases/metabolismo , Helicase da Síndrome de Werner/metabolismoRESUMO
The Hippo pathway controls cell homeostasis, and its deregulation can lead to human diseases. In this pathway, the YAP and TAZ transcriptional cofactors play a key role in stimulating gene transcription through their interaction with the TEAD transcriptional factors. Our study of YAP and TAZ peptides in biochemical and biophysical assays shows that both proteins have essentially the same affinity for TEAD. Molecular modeling and structural biology data suggest that they also bind to the same site on TEAD. However, this apparent similarity hides differences in the ways in which the two proteins interact with TEAD. The secondary structure elements of their TEAD binding site do not contribute equally to the overall affinity, and critical interactions with TEAD are made through different residues. This convergent optimization of the YAP/TAZ TEAD binding site suggests that the similarity in the affinities of binding of YAP to TEAD and of TAZ to TEAD is important for Hippo pathway functionality.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Via de Sinalização Hippo , Humanos , Imuno-Histoquímica , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
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 ß-strand, an α-helix, and an Ω-loop. Earlier results have shown that the ß-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 ß-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 ß-strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that ß-sheet fluctuations play a relevant role in YAP53-57 ß-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 ß-strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity.
Assuntos
Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Fatores de Transcrição/química , Proteínas de Ligação a DNA/química , Conformação Proteica em Folha beta , Regulação da Expressão Gênica , Ligação ProteicaRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Magnésio/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Receptor IGF Tipo 1/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Conformação Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Pirróis/farmacologia , Estaurosporina/farmacologia , Especificidade por SubstratoRESUMO
The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352GlnSd) that is predominantly acylated on the lysine (Lys350Sd). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição de Domínio TEA , Animais , Cisteína/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoilação , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transativadores/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/química , DNA/química , Proteínas de Drosophila/química , Proteínas Intrinsicamente Desordenadas/química , Fatores de Transcrição de Domínio TEA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA/genética , DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Via de Sinalização Hippo/genética , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição de Domínio TEA/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans , Evolução Molecular , Fatores de Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Humanos , Domínios Proteicos , Especificidade da Espécie , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Bases de Dados de Proteínas , Células HEK293 , Humanos , Conformação Proteica , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Musculares/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Animais , Sítios de Ligação , Drosophila , Células HEK293 , Humanos , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição de Domínio TEARESUMO
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 Tyr421HisTEAD1 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 ).
Assuntos
Proteínas de Ciclo Celular/genética , Distrofias Hereditárias da Córnea/genética , Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Mapas de Interação de Proteínas/genética , Degeneração Retiniana/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Aciltransferases , Proteínas de Ciclo Celular/química , Distrofias Hereditárias da Córnea/patologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Mutação/genética , Proteínas Nucleares/genética , Ligação Proteica/genética , Degeneração Retiniana/patologia , Transdução de Sinais/genética , Fatores de Transcrição de Domínio TEARESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Mutação , Ligação Proteica , Conformação Proteica , Termodinâmica , Água/químicaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Mapas de Interação de Proteínas , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas Musculares/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Acilação/fisiologia , Humanos , Modelos Moleculares , Transdução de Sinais/fisiologia , Fatores de Transcrição de Domínio TEA , Transativadores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.
Assuntos
Bioensaio , Descoberta de Drogas , Fluorescência , Termodinâmica , Calorimetria , Cinética , Ligação Proteica/fisiologia , Desnaturação Proteica , Receptor IGF Tipo 1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , TemperaturaRESUMO
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 site-specific 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.