La phosphorylation, par la caséine kinase II, des centrines humaines, régule l'association avec leurs cibles cellulaires

Les centrines sont des calciprotéines caractérisées par quatre motifs EF-hand et leur grande conservation chez les eucaryotes. L interaction des centrines avec plusieurs cibles cellulaires à pour conséquence leur participation, à plusieurs mécanismes cellulaires, comme la cascade de photo transduction dans la rétine, la réparation de l ADN, la duplication du centrosome et le transport de l ARNm du noyau vers le cytoplasme. Les partenaires cellulaires des centrines possèdent un ou plusieurs motifs ayant des résidus hydrophobes conservés dans la position 1, 4 et 8, qui lient la calciprotéine. La régulation des fonctions des centrines est réalisée par le calcium, mais également, par la phosphorylation. Le travail de thèse a porté sur la caractérisation du processus de phosphorylation, in vitro, des centrines humaines, par la caséine kinase II, et sur l étude de l effet de la phosphorylation sur l interaction des centrines avec leurs cibles, tout en gardant une attention spécifique au mécanisme d association des complexes. De plus, une nouvelle cible cellulaire a été analysée dans le cadre de ce projet : la Transducine b, protéine impliquée dans la cascade de photo transduction et caractérisée par un motif de liaison à la centrine situé à son extrémité C-terminale.Centrins are calcium-binding proteins characterized by fours EF-hand motifs and by a good conservation among the eukaryotes. The interaction of centrins with several cellular targets has for consequence the association of this protein to various cellular functions, such as involvement in visual phototransduction cascade, DNA repair, centrosome duplication and the export of ARNm from the nucleus to the cytoplasm. The cellular binding partners of centrins are characterized by one or several repetitive the sequence of which is composed of hydrophobic residues in the 1, 4 and 8 position, responsible for the binding to centrins. The function of centrins is regulated by the conformational changes induced by calcium, as well as, by phosphorylation. The thesis work focused on the in vitro casein kinase II protein-phosphorylation process concerning the centrins, and on the study of the effect of the phosphorylation on the binding properties of centrins with their cellular targets, keeping a deep attention to the mechanisms of association to form the complexes. Furthermore, a new cellular target was analyzed during this project: b-Transducin, a protein involved in the visual phototransduction cascade, which is characterized by one centrin-binding motif situated to its C-terminal extremity.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

CK2 phosphorylation of human centrins 1 and 2 regulates their binding to the DNA repair protein XPC, the centrosomal protein Sfi1 and the phototransduction protein transducin β

Centrins are calcium-binding proteins that can interact with several cellular targets (Sfi1, XPC, Sac3 and transducin β) through the same hydrophobic triad. However, two different orientations of the centrin-binding motif have been observed: W1xxL4xxxL8 for XPC (xeroderma pigmentosum group C protein) and the opposite orientation L8xxxL4xxW1 for Sfi1 (suppressor of fermentation-induced loss of stress resistance protein 1), Sac3 and transducin β. Centrins are also phosphorylated by several protein kinases, among which is CK2. The purpose of this study was to determine the binding mechanism of human centrins to three targets (transducin β, Sfi1 and XPC), and the effects of in vitro phosphorylation by CK2 of centrins 1 and 2 with regard to this binding mechanism. We identified the centrin-binding motif at the COOH extremity of transducin β. Human centrin 1 binds to transducin β only in the presence of calcium with a binding constant lower than the binding constant observed for Sfi1 and for XPC. The affinity constants of centrin 1 were 0.10 106 M−1, 249 106 M−1 and 52.5 106 M−1 for Trd, R17-Sfi1 and P17-XPC respectively. CK2 phosphorylates human centrin 1 at residue T138 and human centrin 2 at residues T138 and S158. Consequently CK2 phosphorylation abolished the binding of centrin 1 to transducin β and reduced the binding to Sfi1 and XPC. CK2 phosphorylation of centrin 2 at T138 and S158 abolished the binding to Sfi1 as assessed using a C-HsCen2 T138D-S158D phosphomimetic form of centrin 2

Diacylglyceride kinases, sphingosine kinases and NAD kinases: distant relatives of 6-phosphofructokinases

International audienceDiacylglyceride kinases, sphingosine kinases, NAD kinases and 6-phosphofructokinases are thought to be related despite large evolution of their sequences. Discovery of a common signature has led to the suggestion that they possess a similar phosphate-donor-binding site and a similar phosphorylation mechanism. The substrate- and allosteric-binding sites are much more divergent and their delineation remains to be determined experimentally

NAD kinases use substrate-assisted catalysis for specific recognition of NAD.

International audienceHere we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg(2+) ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases

α-Peptoïdes et composés apparentés : synthèse et contrôle de la conformation

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Structure, Dynamics and Thermodynamics of the Human Centrin 2/hSfi1 Complex

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the similar to 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -angstrom translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C+HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand. (C) 2009 Elsevier Ltd. All rights reserve

A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin

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8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics