Exploration in silico d'une mini spectrine, l'a-actinine (dynamique et interactions)

La superfamille des spectrines est un groupe important de protéines du cytosquelette. Bien qu'ayant des fonctions très différentes, les membres de cette famille partagent un niveau surprenant d'homologie structurale, et sont composés de domaines conservés tels que les répétitions spectrines, les domaines CH de liaison à l'actine et les mains EF. Jusqu'à présent, des études ont porté sur les propriétés mécaniques de 2 à 3 unités répétitives de la spectrine ou sur le "rod domain" de l'alpha-actinine, sans tenir compte du rôle potentiel des domaines globulaires. Dans ces travaux de thèse, la mécanique du modèle de l'alpha-actinine entière a été examinée. Dans un premier temps, une étude hiérarchique par analyse de modes normaux du domaine central de l'alpha-actinine a été réalisée afin d'évaluer l'effet de l'organisation 3D des répétitions sur la mécanique du système. Le développement d'un outil d'analyse spécifique a permis de caractériser finement la nature des mouvements. L'impact des domaines globulaires sur ces propriétés mécaniques a aussi été exploré. Dans un second temps, l'influence de la concentration ionique sur la flexibilité du "rod domain" a été examinée. Nous montrons ainsi que : les caractéristiques structurales du "rod domain" lui confèrent ses propriétés mécaniques uniques ; les domaines globulaires modifient le comportement mécanique de la fibre ; l'écrantage des charges à la surface du "rod domain" par les ions modifie sa flexibilité. Cette étude ouvre la voie vers l'extrapolation des caractéristiques mécaniques de l'alpha-actinine aux autres membres de la superfamille des spectrines.The spectrin superfamilly is a large group of cytoskeletal proteins. Although they have very different functions, the members of this family share a surprising level of structural homology and are composed of conserved domains such a spectrin repeats and globular actin binding domains and EF hands. So far, studies have focused on the mechanical properties of 2 to 3 repetitive units of spectrin or the rod domain of the alpha-actinin, regardless the potential role of the globular domains. In this thesis work, the mechanical model of the entire alpha-actinin was examined. Initially, a hierarchical study by normal modes analysis of the central domain of the alpha-actinin was performed to evaluate the effect of the 3D organization of the repeats on the mechanics of the system. The development of a specific analysis tool allowed us to finely characterize the nature of movements. The impact of the globular domains on these mechanical properties was also explored. In the second part of this work, the influence of ion concentration on the flexibility of the rod domain was discussed. We thus show that : the structural characteristics of the rod domain give it its unique mechanical properties, and the globular domains alter the mechanical behaviour of the fibre ; the screening of charges on the surface of the rod domain by ions changes its flexibility. This study opens the way to the extrapolation of the mechanical properties of alpha-actinin to the other members of the superfamily of spectrin.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Selection of active site conformations for virtual screening.

<p>(A) Four of the 49 conformations defining the path were selected for virtual screening. Protein secondary structures are shown schematically as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060955#pone-0060955-g002" target="_blank">Figure 2</a>. Transparent green spheres show the enclosed void volume of the pocket, with the ligand inside the first three structures (opaque green). (B) Structure of the two identified novel inhibitors of <i>Tc</i>PRAC.</p

Effects of <i>Tc</i>PRAC inhibition by OxoPA and BrOxoPA on parasite interaction with host cells in vitro.

<p>Vero cells cultures were infected for 17 h at 37°C with cultured trypomastigotes at a 10∶1 parasite/cell ratio then washed and incubated for 17 h with 0, 100 and 1000 µM of PYC, or 0, 10 and 30 µM OxoPA or Br-OxoPA in fresh medium. Total parasite numbers/culture were estimated by capture ELISA.</p

Kinetics of D-proline formation with time in the presence or absence of <i>Tc</i>PRAC inhibitors.

<p>(A) Concentrations of D-proline formed were determined by polarimetry in reaction assays containing 0.15 µM of <i>Tc</i>PRAC and 40 mM L proline (see Material and Methods). Optical rotation was measured every 10 s for 8 min, with 10 µM PYC competitive inhibitor (black circles), 10 µM OxoPA (black squares) and 10 µM Br-OxoPA (black triangles), or without inhibitor (white circles). (B) Percentage of residual activity after pre-incubation of the enzyme with different concentrations of OxoPA and BrOxoPA for 5 minutes (upper panel) and 10 minutes (lower panel).</p

Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.

<p>Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.</p

New inhibitors induce a decrease of cellular uptake of <i>T. cruzi</i>.

<p>New inhibitors induce a decrease of cellular uptake of <i>T. cruzi</i>.</p

Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.

<p>Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.</p

Transition path characteristics.

<p>(A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. Transition path characteristics. (A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. (C) Cavity volume and extension in transition path intermediates, and docked molecules properties. Volume and extension are calculated as explained in Material and Methods. The volume is displayed by the thin line curve. Cavity extension is displayed by the thick curve and crosses mark intermediates that were used for virtual screening. The extension of PYC is shown by the horizontal line, that of OxoPA in all-trans conformation is shown by the horizontal dashed line. The extension of BrOxoPA in all-trans conformation is given in dotted line for two extreme rotamers on the C4–C5 bond. The average molecular weight of the library compounds successfully docked in conformers 1, 4 and 10 is displayed by filled circles. For clarity, the average mass has been divided by 2 to fit the same scale as the cavity volume.</p

Structure of initial compounds and analogues.

<p>(1) PYC and its nitrogen-bearing analogues: (2) Imidazole-4-carboxylic acid, (3) 1-<i>H</i>-Imidazole-2-carboxylic acid and (4a) PZC, (4b) Cl-PZC and (4c) Br-PZC.</p