Vincent Villeret

University of Lille Nord de France, Lille, Nord-Pas-de-Calais, France

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Publications (80)395.94 Total impact

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    ABSTRACT: MED26 is a subunit of the Mediator, a very large complex involved in regulation of gene transcription by RNA Polymerase II. MED26 regulates the switch between initiation and elongation phases of the transcription. This function requires interaction of its N-terminal domain (NTD) with several protein partners implicated in transcriptional regulation. Molecular details of the structure and interaction mode of MED26 NTD would improve understanding of this complex regulation. As a first step towards structural characterization, sequence specific (1)H, (13)C and (15)N assignments for MED26 NTD was performed based on Nuclear Magnetic Resonance spectroscopy. TALOS+ analysis of the chemical shifts data revealed a domain solely composed of helices. Assignments will be further used to solve NMR structure and dynamics of MED26 NTD and investigate the molecular details of its interaction with protein partners.
    No preview · Article · Feb 2016 · Biomolecular NMR Assignments
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    Preview · Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
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    ABSTRACT: The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Preview · Article · Jun 2015 · Nucleic Acids Research
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    ABSTRACT: Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded β-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved 'lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1.
    Full-text · Article · Jun 2015 · Nature Communications
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    ABSTRACT: Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.
    Full-text · Article · Feb 2015 · PLoS Pathogens
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    ABSTRACT: Many bacterial pathogens use Type Three Secretion System (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens suggesting a common mechanism of recognition and secretion of effectors. On the contrary no common motif is yet identified for effectors allowing T3SS recognition. In this work we performed a biochemical and structural characterization of Salmonella SopB/SigE chaperone/effector complex by SAXS. Our results showed that the SigD/SigE complex is assembled in dynamic homo-hexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome including ATPase hexameric ring; this organization suggests that this structural feature is important for the ATPase recognition by T3SS. Our work constitutes the first evidence in solution of the oligomerization of an effector analogous to the organization of the secretion machinery. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Full-text · Article · Nov 2014 · Journal of Bacteriology
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    ABSTRACT: Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120-160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.
    Full-text · Article · Jul 2014 · Biophysical Journal
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    ABSTRACT: Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide, and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein we have developed fragment-based approaches. In the current study we combined surface plasmon resonance assay, X-ray crystallography, ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, in vitro and ex vivo activities of these compounds will be discussed.
    Full-text · Article · May 2014 · Journal of Medicinal Chemistry
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    Full-text · Dataset · Dec 2013
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    ABSTRACT: Haemophilus influenzae HxuA is a cell-surface protein with haem-haemopexin binding activity which is key to haem acquisition from haemopexin and thus is one of the potential sources of haem for this microorganism. HxuA is secreted by its specific transporter HxuB. HxuA/HxuB belongs to the so-called two-partner secretion systems (TPSs) that are characterized by a conserved N-terminal domain in the secreted protein which is essential for secretion. Here, the 1.5 Å resolution structure of the secretion domain of HxuA, HxuA301, is reported. The structure reveals that HxuA301 folds into a β-helix domain with two extra-helical motifs, a four-stranded β-sheet and an N-terminal cap. Comparisons with other structures of TpsA secretion domains are reported. They reveal that despite limited sequence identity, strong structural similarities are found between the β-helix motifs, consistent with the idea that the TPS domain plays a role not only in the interaction with the specific TpsB partners but also as the scaffold initiating progressive folding of the TpsA proteins at the bacterial surface.
    No preview · Article · Dec 2013 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: 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 of 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.
    Full-text · Article · Sep 2013 · Biochimica et Biophysica Acta
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    ABSTRACT: Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.
    Full-text · Article · Jul 2013 · Molecular Microbiology
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    ABSTRACT: Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA-binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.
    Full-text · Article · May 2013 · PLoS Genetics
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    ABSTRACT: Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.
    Full-text · Article · May 2013 · PLoS Genetics
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    ABSTRACT: PEA3, 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.
    Full-text · Article · Mar 2013 · Nucleic Acids Research
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    Dataset: Figure S3
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    ABSTRACT: Effect of PARP-1 catalytic inhibition on the level of Ets-1 and γH2AX. MDA-MB-231 cells were treated with PJ-34 (1 µM), 5-AIQ (1 µM) or ABT-888 (1 µM) for 20 h. Cell lysates (30 µg total proteins) were analysed by Western blot using different antibodies (see Materials and Methods) against Ets-1, γH2AX, p53 and β-Actin. (TIF)
    Preview · Dataset · Feb 2013
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    Dataset: Figure S1
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    ABSTRACT: MALDI-TOF spectrum of PARP-1 purified by streptavidin pull-down. (TIF)
    Preview · Dataset · Feb 2013
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    ABSTRACT: Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.
    Preview · Article · Feb 2013 · PLoS ONE
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    Dataset: Figure S2
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    ABSTRACT: Sub-localisation of Ets-1 and PARP-1 in MDA-MB-231 cells by immunofluorescence. Ets-1 is visualised in red (Alexa Fluor® 594), and PARP-1 in green (Alexa Fluor® 388). The insets are close-ups of the boxed cells. Nuclei were visualised using DAPI stain. Cells were examined under a fluorescence microscope at ×40 magnification. Scale bar = 20 µm. (TIF)
    Preview · Dataset · Feb 2013
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    Dataset: Figure S6
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    ABSTRACT: Determination of γH2AX-positive cells for statistical analyses. γH2AX-positive cells were determined by counting γH2AX foci, visualised here in red (Alexa Fluor® 594), in the cell nucleus from immunofluorescence experiments. Cells with no or less than 10 γH2AX foci were considered to be negative (γH2AX −; 1 and 2); while cells with more than 10 γH2AX foci were considered to be positive (γH2AX +; 3 and 4). (TIF)
    Preview · Dataset · Feb 2013

Publication Stats

2k Citations
395.94 Total Impact Points

Institutions

  • 2009-2015
    • University of Lille Nord de France
      Lille, Nord-Pas-de-Calais, France
  • 2004-2015
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2007-2011
    • Université du Droit et de la Santé Lille 2
      Lille, Nord-Pas-de-Calais, France
    • Université des Sciences et Technologies de Lille 1
      Lille, Nord-Pas-de-Calais, France
  • 2004-2008
    • Institut Pasteur de Lille
      Lille, Nord-Pas-de-Calais, France
  • 1997-2008
    • Ghent University
      • Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE)
      Gent, VLG, Belgium
  • 2005-2007
    • Institut de Biologie de Lille
      Lille, Nord-Pas-de-Calais, France
  • 1994-1996
    • University of Liège
      Luik, Walloon Region, Belgium
  • 1995
    • Harvard University
      • Department of Chemistry and Chemical Biology
      Cambridge, Massachusetts, United States