Molecular Recognition Unit

Brussels, Belgium

Molecular Recognition Unit

Brussels, Belgium
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Hadzi S.,Vrije Universiteit Brussel | Hadzi S.,Molecular Recognition Unit | Hadzi S.,University of Ljubljana | Garcia-Pino A.,Vrije Universiteit Brussel | And 8 more authors.
Nucleic Acids Research | Year: 2017

Toxin-antitoxin (TA) modules are small operons involved in bacterial stress response and persistence. higBA operons form a family of TA modules with an inverted gene organization and a toxin belonging to the RelE/ParE superfamily. Here, we present the crystal structures of chromosomally encoded Vibrio cholerae antitoxin (VcHigA2), toxin (VcHigB2) and their complex, which show significant differences in structure and mechanisms of function compared to the higBA module from plasmid Rts1, the defining member of the family. The VcHigB2 is more closely related to Escherichia coli RelE both in terms of overall structure and the organization of its active site. VcHigB2 is neutralized by VcHigA2, a modular protein with an N-terminal intrinsically disordered toxin-neutralizing segment followed by a C-terminal helix-turn-helix dimerization and DNA binding domain. VcHigA2 binds VcHigB2 with picomolar affinity, which is mainly a consequence of entropically favorable de-solvation of a large hydrophobic binding interface and enthalpically favorable folding of the N-terminal domain into an α-helix followed by a ß-strand. This interaction displaces helix α3 of VcHigB2 and at the same time induces a one-residue shift in the register of ß-strand ß3, thereby flipping the catalytically important Arg64 out of the active site. © The Author(s) 2017.

Burger V.M.,Massachusetts Institute of Technology | Vandervelde A.,Vrije Universiteit Brussel | Vandervelde A.,Molecular Recognition Unit | Hendrix J.,Catholic University of Leuven | And 7 more authors.
Journal of the American Chemical Society | Year: 2017

The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin protein CcdA is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm. Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA gyrase and thereby averts cell death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states, a closed state and an open state, that are adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Förster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdA's cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell fate. © 2017 American Chemical Society.

Drobnak I.,University of Ljubljana | Drobnak I.,University of Notre Dame | De Jonge N.,Molecular Recognition Unit | De Jonge N.,Vrije Universiteit Brussel | And 7 more authors.
Journal of the American Chemical Society | Year: 2013

Intrinsically disordered proteins (IDPs) are proteins that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into a defined structure when interacting with specific binding partners. The energetic implications of such behavior have been widely discussed, yet experimental thermodynamic data is scarce. We present here a thorough thermodynamic and structural study of the binding of an IDP (antitoxin CcdA) to its molecular target (gyrase poison CcdB). We show that the binding-coupled folding of CcdA is driven by a combination of specific intramolecular interactions that favor the final folded structure and a less specific set of intermolecular contacts that provide a desolvation entropy boost. The folded structure of the bound IDP appears to be defined largely by its own amino acid sequence, with the binding partner functioning more as a facilitator than a mold to conform to. On the other hand, specific intermolecular interactions do increase the binding affinity up to the picomolar range. Overall, this study shows how an IDP can achieve very strong and structurally well-defined binding and it provides significant insight into the molecular forces that enable such binding properties. © 2013 American Chemical Society.

Ghequire M.G.K.,Catholic University of Leuven | Loris R.,Molecular Recognition Unit | Loris R.,Free University of Brussels | De Mot R.,Catholic University of Leuven
Biochemical Society Transactions | Year: 2012

Arguably, bacteriocins deployed inwarfare among related bacteria are among themost diverse proteinacous compounds with respect to structure and mode of action. Identification of the first prokaryotic member of the so-called MMBLs (monocot mannose-binding lectins) or GNA (Galanthus nivalis agglutinin) lectin family and discovery of its genus-specific killer activity in the Gram-negative bacteria Pseudomonas and Xanthomonas has added yet another kind of toxin to this group of allelopathic molecules. This novel feature is reminiscent of the protective function, on the basis of antifungal, insecticidal, nematicidal or antiviral activity, assigned to or proposed for several of the eukaryotic MMBL proteins that are ubiquitously distributed among monocot plants, but also occur in some other plants, fish, sponges, amoebae and fungi. Direct bactericidal activity can also be effected by a C-type lectin, but this is a mammalian protein that limits mucosal colonization by Grampositive bacteria. The presence of two divergent MMBL domains in the novel bacteriocins raises questions about task distribution between modules and the possible role of carbohydrate binding in the specificity of target strain recognition and killing. Notably, bacteriocin activity was also demonstrated for a hybrid MMBL protein with an accessory protease-like domain. This association with one or more additional modules, often with predicted peptide-hydrolysing or -binding activity, suggests that additional bacteriotoxic proteins may be found among the diverse chimaeric MMBL proteins encoded in prokaryotic genomes. A phylogenetic survey of the bacterial MMBL modules reveals a mosaic pattern of strongly diverged sequences, mainly occurring in soil-dwelling and rhizosphere bacteria, which may reflect a trans-kingdom acquisition of the ancestral genes. ©The Authors Journal compilation ©2012 Biochemical Society.

Ghequire M.G.K.,Catholic University of Leuven | Li W.,Catholic University of Leuven | Proost P.,Rega Institute for Medical Research | Loris R.,Molecular Recognition Unit | And 2 more authors.
Environmental Microbiology Reports | Year: 2012

Summary: The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv. malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin (llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1 and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified recombinant proteins, LlpAPss642 and LlpAXcm761, display genus-specific antibacterial activity across species boundaries. The inhibitory spectrum of the P.syringae bacteriocin overlaps partially with those of the P.putida and P.fluorescens LlpAs. Notably, Xanthomonas axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761. The functional characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and suggests its involvement in competition among closely related plant-associated bacteria with different lifestyles. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Volkov A.N.,Vrije Universiteit Brussel | Volkov A.N.,Molecular Recognition Unit | Wohlkonig A.,JAST Laboratory | Soror S.H.,JAST Laboratory | And 3 more authors.
Biochemistry | Year: 2013

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [2H, 13C,15N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy. © 2013 American Chemical Society.

Ghequire M.G.K.,Catholic University of Leuven | Garcia-Pino A.,Molecular Recognition Unit | Garcia-Pino A.,Vrije Universiteit Brussel | Lebbe E.K.M.,Catholic University of Leuven | And 4 more authors.
PLoS Pathogens | Year: 2013

Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity. © 2013 Ghequire et al.

Gelens L.,Vrije Universiteit Brussel | Hill L.,Vrije Universiteit Brussel | Hill L.,Molecular Recognition Unit | Vandervelde A.,Molecular Recognition Unit | And 4 more authors.
PLoS Computational Biology | Year: 2013

Toxin-Antitoxin modules are small operons involved in stress response and persister cell formation that encode a "toxin" and its corresponding neutralizing "antitoxin". Regulation of these modules involves a complex mechanism known as conditional cooperativity, which is supposed to prevent unwanted toxin activation. Here we develop mathematical models for their regulation, based on published molecular and structural data, and parameterized using experimental data for F-plasmid ccdAB, bacteriophage P1 phd/doc and E. coli relBE. We show that the level of free toxin in the cell is mainly controlled through toxin sequestration in toxin-antitoxin complexes of various stoichiometry rather than by gene regulation. If the toxin translation rate exceeds twice the antitoxin translation rate, toxins accumulate in all cells. Conditional cooperativity and increasing the number of binding sites on the operator serves to reduce the metabolic burden of the cell by reducing the total amounts of proteins produced. Combining conditional cooperativity and bridging of antitoxins by toxins when bound to their operator sites allows creation of persister cells through rare, extreme stochastic spikes in the free toxin level. The amplitude of these spikes determines the duration of the persister state. Finally, increases in the antitoxin degradation rate and decreases in the bacterial growth rate cause a rise in the amount of persisters during nutritional stress. © 2013 Gelens et al.

Loris R.,Molecular Recognition Unit | Loris R.,Vrije Universiteit Brussel | Garcia-Pino A.,Molecular Recognition Unit | Garcia-Pino A.,Vrije Universiteit Brussel
Chemical Reviews | Year: 2014

Originally identified as plasmid-stabilizing entities, toxin-antitoxin (TA) modules are ubiquitous in the genomes of prokaryotes and archeae. Most commonly they constitute small operons that encode two genes. The downstream gene encodes for a toxic protein, while the upstream 'antitoxin' gene protects the cell against this toxin. TA antitoxins typically contain a significant amount of intrinsic disorder, with variable degrees of prestructuring. The main function of the IDP segments is folding-upon-binding, and this functionality is directly linked to the evolutionary origin of TA modules. Therefore, in order to understand the nature and functionality of intrinsic disorder in antitoxins, one first has to look at the architectures of type II TA modules and how they likely evolved. One may consider a regulatory segment breaking off from a toxin precursor and fusing to a common transcription regulator domain, thus creating a module with novel regulatory properties.

Van De Water K.,Vrije Universiteit Brussel | Van De Water K.,Molecular Recognition Unit | Van Nuland N.A.J.,Vrije Universiteit Brussel | Van Nuland N.A.J.,Molecular Recognition Unit | And 2 more authors.
Chemical Science | Year: 2014

Invisible to most biophysical techniques, transient intermediates formed during biomolecular association orchestrate protein recognition and binding. Here, we study such minor species mediating the interaction between physiological partners, cytochrome c and cytochrome c peroxidase, by paramagnetic relaxation enhancement NMR spectroscopy. The visualization of multiple protein-protein orientations constituting the transient encounter state reveals a broad spatial distribution, which is in striking agreement with that obtained in earlier theoretical simulations. Being inactive in the intermolecular electron transfer, the encounter complex pre-orients the interacting molecules, enabling a reduced dimensionality search of the dominant, functionally active bound form. The encounter complex is insensitive to the redox and spin states of the interacting molecules, suggesting that its properties are determined by protein polypeptides rather than heme cofactors. © the Partner Organisations 2014.

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