The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members

International audiencePEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro . Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Sumo et le désordre structural font-ils bon ménage ?

La sumoylation représente, après l’ubiquitination, l’exemple le plus étudié de modification post-traductionnelle impliquant la liaison d’une protéine à une autre. Cependant, alors que l’ubiquitination est impliquée principalement dans la dégradation des protéines par le protéasome, la sumoylation semble réguler les propriétés biochimiques de ses substrats (localisation cellulaire, interaction protéique, activité, …). Pour venir lier une protéine appelée Sumo (Small Ubiquitin-like Modifier) sur un substrat, la sumoylation emprunte une voie enzymatique analogue à celle de l’ubiquitination mais utilise des enzymes différentes. A ce jour, bien que plusieurs centaines de substrats de la sumoylation aient été identifiés, seules 5 structures de protéines sumoylées ont été résolues. Elles ne sont vraisemblablement pas représentatives de l’ensemble des substrats de la sumoylation et mon travail de thèse vise à élargir les connaissances structurales sur la sumoylation pour permettre de dégager des concepts généraux. <p>Les études sur la sumoylation se heurtent généralement à la difficulté d’obtenir les substrats sumoylés. Ce projet a donc nécessité, au niveau technique, la mise au point d’un système de sumoylation in vivo en bactérie permettant de modifier des quantités importantes de protéines et de les purifier efficacement. <p>Des analyses bioinformatiques nous ont permis d’identifier des substrats de la sumoylation propices à une étude structurale de leur forme sumoylée. Au terme de ces analyses, nous avons retenu 3 protéines :DJ-1, PPARγ et IκBα. Bien que la complexité du sujet nous ait ensuite amené à écarter DJ-1 et PPARγ, nous sommes parvenus à purifier la forme sumoylée d’IκBα. Ce résultat nous a permis d’entreprendre une campagne de cristallogenèse d’IκBα complexé au facteur de transcription NF-κB. L’obtention d’IκBα sumoylé permettra également d’aborder des études fonctionnelles pour améliorer la compréhension du rôle de la sumoylation de ce substrat. <p>Nos analyses bioinformatiques ont également révélé que dans plus de 60% des cas, les sites de sumoylation des substrats se trouvent dans des zones prédites intrinsèquement désordonnées. L’importance du désordre dans le processus de sumoylation était jusqu’alors largement sous-estimée. A titre d’exemple, nous avons étudié par diffusion des rayons X aux petits angles la structure du domaine transactivateur du facteur de transcription ERM sous forme non modifiée et sous forme sumoylée. Cette étude indique que la sumoylation d’ERM n’induit pas le repliement de ce domaine transactivateur. De même, il apparait peu probable, au vu de la flexibilité de cette région, que la sumoylation empêche des interactions avec certains partenaires cellulaires. Dans ce contexte, la sumoylation semble servir de plateforme de recrutement de partenaires, reconnaissant de manière spécifique le Sumo. Ce mécanisme pourrait se généraliser à l’ensemble des sites de sumoylation prédits dans des zones intrinsèquement désordonnées. <p>Le système de sumoylation que nous avons développé permet de produire des protéines sumoylées pures en grande quantité et pourra également servir à identifier des protéines reconnaissant spécifiquement les substrats modifiés. Tous ces éléments devraient permettre de progresser dans la compréhension de cette modification post-traductionnelle impliquée dans de nombreux processus cellulaires fondamentaux. <p>Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Purification of SUMO-1 modified IκBα and complex formation with NF-κB.

Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available. Purification of sumoylated substrates in vitro or in bacteria have been previously reported but separating the sumoylated protein from the undesired unmodified fraction is often technically challenging, inefficient and time consuming. Here we develop a new vector system for in vivo sumoylation in Escherichia coli which improves purification of sumoylated proteins. We describe the purification of IκBα, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex IκBα-SUMO-1/NF-κB. After a first GST affinity chromatography and GST-tag removal, a unique metal-ion affinity chromatography using a 6xHis-SUMO-1 tag results in mgs of highly pure SUMO-1 modified IκBα. Our pure SUMO-1 modified IκB/NF-κB complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified IκBα form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Crystal structure of human Mediator subunit MED23

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The structural organization of the N-terminus domain of SopB, a virulence factor of Salmonella, depends on the nature of its protein partners

The TTSS is used by Salmonella and many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Once translocated these so-called effector proteins hijack a vast array of crucial cellular functions to the benefit of the bacteria. In the bacterial cytoplasm, some effectors are stabilized and maintained in a secretion competent state by interaction with specific type III chaperones. In this work we studied the conformation of the Chaperone Binding Domain of the effector named Salmonella Outer protein B (SopB) alone and in complex with its cognate chaperone SigE by a combination of biochemical, biophysical and structural approaches. Our results show that the N-terminus part of SopB is mainly composed by α-helices and unfolded regions whose organization/stabilization depends on their interaction with the different partners. This suggests that the partially unfolded state of this N-terminal region, which confers the adaptability of the effector to bind very different partners during the infection cycle, allows the bacteria to modulate numerous host cells functions limiting the number of translocated effectors. © 2013 Elsevier B.V.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Exploring drug target flexibility using in situ click chemistry: Application to a mycobacterial transcriptional regulator

In situ click chemistry has been successfully applied to probe the ligand binding domain of EthR, a mycobacterial transcriptional regulator known to control the sensitivity of Mycobacterium tuberculosis to several antibiotics. Specific protein-templated ligands were generated in situ from one azide and six clusters of 10 acetylenic fragments. Comparative X-ray structures of EthR complexed with either clicked ligand BDM14950 or its azide precursor showed ligand-dependent conformational impacts on the protein architecture. This approach revealed two mobile phenylalanine residues that control the access to a previously hidden hydrophobic pocket that can be further exploited for the development of structurally diverse EthR inhibitors. This report shows that protein-directed in situ chemistry allows medicinal chemists to explore the conformational space of a ligand-binding pocket and is thus a valuable tool to guide drug design in the complex path of hit-to-lead processes. © 2010 American Chemical Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25

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Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

NMR structure of the human Mediator MED25 ACID domain.

International audienceMED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed β-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel, defined by strands B4-B7-B6