Probing Structural and Motional Features of the C-Terminal Part of the Human Centrin 2/P17-XPC Microcrystalline Complex by Solid-State NMR Spectroscopy

International audienceAn insight into structural and motional features of the C-terminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine not only the quality of solid-state NMR spectra but can also dramatically modify the dynamic state of water molecules and the internal mobility of the protein. Two-dimensional (2D) 13C - 13C and 15N - 15N correlation spectra reveal intra- and inter-residue dipolar connectivities and provide partial, site-specific assignments of 13C and 15N resonance signals. The secondary structure of the C-ter HsCen2 /P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca2+ cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC

iShoes for blind and visually impaired people

This paper presents the development of an iShoes system for blind and visually impaired people. The iShoes system utilizes a microcontroller with sound output interfaced with ultrasonic sensors. The prototype system is designed to be specifically mounted on/in the shoes to aid navigation in urban routes. The ultrasonic transducers determine the range from an obstacle and then play an audio message to reflect the distance from the target. This system will assist blind and visually impaired people in navigating a path through an unfamiliar environment. Keywords—Ultrasonic guidance system; ultrasonic sensors; blind, visually impaired; navigatio

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

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