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See detailMetal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
Laurent, Clémentine ULg; Lekeux, Gilles ULg; Ukuwela, Ashwinie A et al

in Plant Molecular Biology (2016), 90

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding ... [more ▼]

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. <br />Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. <br />The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. <br />Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases. [less ▲]

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See detailInvestigation of a suitable in vitro dissolution test for itraconazole-based solid dispersions
Thiry, Justine ULg; Broze, Guy ULg; Pestieau, Aude ULg et al

in European Journal of Pharmaceutical Sciences (2016)

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See detailProtein structure modeling using backbone chemical shifts
Wanko Nembot, Alexis Marius ULg; Damblon, Christian ULg

Poster (2015, December 01)

The knowledge of the tridimensional structure of a protein is essential to study its interactions and understand its mode of action. The Purpose of our work is to quickly and easily determine the ... [more ▼]

The knowledge of the tridimensional structure of a protein is essential to study its interactions and understand its mode of action. The Purpose of our work is to quickly and easily determine the structure of proteins using the backbone chemical shifts. Backbone chemical shifts data are NMR parameters that can be rapidly, easily and accurately measured. This parameter is very sensitive to the conformation of amino acids and is used to deduct the secondary structure (TALOS, RCI,...). We therefore plan to use backbone chemical shifts as constraints on dihedral angles to quickly and easily determine protein structure. Several « de novo » methods like CS-Rosetta , CS23D et CHESHIRE have been recently developed in this purpose. We will use proteins of different sizes for which, the structure (X-ray or NMR structure) and chemical shifts backbone are available for testing the three softwares. Knowing that each of these softwares predicted a large number of low energy models on the one hand, and that the deployment and use of these tools constitutes obstacles for users who are not experts in computer science on the other hand, our goal will be to develop a platform that can easily compare these three methods based on quality of the structure produced. [less ▲]

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See detailRecyclable shape-memory materials based on photo or thermo-reversible crosslinking
Defize, Thomas ULg; Riva, Raphaël ULg; Wauters, Céline et al

Poster (2014, November 11)

Shape memory polymers (SMPs) are remarkable materials able to switch from a stressed deformed state (temporary shape) to their initial relaxed state (permanent shape) by the application of a stimulus ... [more ▼]

Shape memory polymers (SMPs) are remarkable materials able to switch from a stressed deformed state (temporary shape) to their initial relaxed state (permanent shape) by the application of a stimulus, such as heat or light. Typically, the shape memory property is generally observed for chemically or physically cross-linked polymers that exhibit an elastomeric behavior above a phase transition, e.g. glass or melting transition. Cross-linked semi-crystalline poly(ε-caprolactone) (PCL) is already widely studied for the development of SMPs. However, the tensile strength of standard PCL-based SMPs remains quite low, limiting their use in some applications. A convenient way to enhance the strength of SMPs relies on the introduction of nanofillers, such as silica nanoparticles, leading to an enhancement of mechanical strength. Moreover, silica nanoparticles can be advantageously used as multifunctional crosslinking nodes, with the purpose to increase the cross-linking density of the material. As most of SMPs are irreversibly cross-linked material, their reprocessing is impossible preventing any recycling. Thereby, reversible reactions, allowing the formation/cleavage of the network, raise tremendous interest in macromolecular engineering. Recently, a reversibly cross-linked 4-arm star-shaped PCL-based SMP was prepared using the Diels-Alder (DA) reaction between furan and maleimide moieties, well-known to create reversible bonds. This shape memory material demonstrated to be implementable, and so recyclable, and was characterized by excellent fixity and recovery before and after recycling experiments. However, the relatively low retro-DA temperature of the furan- maleimide adducts led to an inelastic deformation during shape memory tensile cycles. In order to get rid of this drawback, two alternative approaches were investigated. Firstly, the substitution of the DA reaction by a photo-reversible reaction, typically the photo- induced (2+2) cycloaddition of coumarins, was proposed to crosslink the PCL matrix. The second approach is based on the use of surface functionalized silica nanoparticles as crosslinking nodes with the purpose to increase the crosslinking density of the material. The network formation and cleavage were studied by solid-state NMR and rheology.4 The resulting shape memory materials were characterized by excellent one-way and two-way shape memory properties as demonstrated by dynamical mechanical analysis. [less ▲]

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See detailStructural and functional analysis of the HMA4 protein
Lekeux, Gilles ULg; Laurent, Clémentine ULg; Damblon, Christian ULg et al

Poster (2014, September 09)

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See detailA variety of roles for versatile zinc in metallo-b-lactamases
Karsisiotis, Andreas Ioannis; Damblon, Christian ULg; Roberts, Gordon C K

in Metallomics (2014)

Metallo-b-lactamases are important as a major source of resistance of pathogenic bacteria to the widely used b-lactam antibiotics. They show considerable diversity in terms of sequence and are grouped ... [more ▼]

Metallo-b-lactamases are important as a major source of resistance of pathogenic bacteria to the widely used b-lactam antibiotics. They show considerable diversity in terms of sequence and are grouped into three subclasses, B1, B2 and B3, which share a common overall fold. In each case the active enzyme has binding sites for two zinc ions in close proximity, although the amino-acid residues which coordinate the metals vary from one subclass to another. In subclasses B1 and B3, there has been controversy about whether both zinc ions are required for activity, but the most recent evidence indicates that there is positive cooperativity in zinc binding and that the catalytically relevant species is the di-zinc enzyme. Subclass B2 enzymes, on the other hand, are active in the mono-zinc state and are inhibited by the binding of a second zinc ion. Evidence for the importance of the zinc ions in substrate binding has come from structures of product complexes which indicate that the b-lactam core binds to subclass B1 and B3 enzymes in a rather consistent fashion, interactions with the zinc ions being centrally important. The zinc ions play key roles in the catalytic mechanism, including facilitating nucleophilic attack on the amide carbonyl by the zinc-bound hydroxide ion, stabilising the anionic tetrahedral intermediate and coordinating the departing amine nitrogen. [less ▲]

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See detailComplete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-b-lactamase and its complex with the inhibitor R-thiomandelic acid
Karsisiotis, Andreas Ioannis; Damblon, Christian ULg; Roberts, Gordon C K

in Biomolecular NMR Assignments (2014), 8

b-Lactamases inactivate b-lactam antibiotics by hydrolysis of their endocyclic b-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-b-lactamase ... [more ▼]

b-Lactamases inactivate b-lactam antibiotics by hydrolysis of their endocyclic b-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-b-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all b-lactam antibiotics. We present here essentially complete ([96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-b-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-b-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-b-lactamase inhibitors. [less ▲]

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See detailSynthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A
Gahungu; Arguelles Arias, Anthony ULg; Fickers, Patrick ULg et al

in Bioorganic & Medicinal Chemistry (2013), 21

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See detailSolution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid
Karsisiotis, Andreas Ioannis; Damblon, Christian ULg; Roberts, Gordon C K

in Biochemical Journal (2013), 456

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present ... [more ▼]

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase BcII and of its complex with R-thiomandelic acid, a broadspectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3–β4 loop. Comparisons with other metallo-β- lactamases highlight the roles of individual amino-acid residues in the active site and the β3–β4 loop in inhibitor binding and provide information on the basis of structure–activity relationships among metallo-β-lactamase inhibitors. [less ▲]

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See detailSynthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.
Gahungu, Mathias; Arguelles-Arias, Anthony; Fickers, Patrick et al

in Bioorganic & Medicinal Chemistry (2013), 21(17), 4958-67

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential ... [more ▼]

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi. [less ▲]

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See detailLigand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry
Tavel, Laurette ULg; Jaquillard, Lucie; Karsisiotis, Andreas et al

in PLoS ONE (2012), 7(4): e36187

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and ... [more ▼]

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1. [less ▲]

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See detailEfficient determination of diffusion coefficients by monitoring transport during recovery delays in NMR
Augustyniak, Rafal; Ferrage, Fabien; Damblon, Christian ULg et al

in Chemical Communications (2012), 48

Detailed reference viewed: 17 (0 ULg)