Ce projet de recherche est focalisé sur le lien entre la structure des molécules odorantes et leurs interactions avec les récepteurs olfactifs exprimés dans les neurones olfactifs. Cette recherche fondamentale est d'une importance primordiale pour la construction d'un «nez virtuel », physiologiquement inspiré, qui reproduit la fonction des 400 types de récepteurs olfactifs impliqués dans la détection des odeurs. Ici, chaque récepteur olfactif est représenté par un système moléculaire qui est reproduit atome par atome dans un modèle informatique. Un protocole optimal a été conçu pour prédire les structures de ces récepteurs grâce à l’analyse bioinformatiques de leurs séquences sous contraintes de données expérimentales. Ensuite, les bases de la relation entre la séquence d’un récepteur et son mécanisme d’activation en fonction de la structure d’une molécule odorante liée à sa cavité ont été établies. Par ailleurs, l’analyse des structures de molécules d’une même famille olfactive peut conduire à l’identification des récepteurs impliqués dans leur perception. L’ensemble de ces résultats constitue les bases pour l’étude des relations structure-odeur à l’ère post-génomique. / This research project is focused on the link between chemical structures of odorant molecules and their interactions with odorant receptors expressed in olfactory neurons. This basic research is of primary importance for building a physiologically-inspired “computational nose” that reproduces the function of the 400 types of odorant receptors involved in the perception of smells. Here, each odorant receptor is represented as a molecular system, reproduced atom per atom in a computational model. An optimal protocol has been built to predict the structure of these receptors using bioinformatics analyses of their sequences under the constraints of experimental data. Then, the relationship between the sequence and the activation mechanism of a receptor as a function of the structure of a molecule bound to its cavity has been established. Furthermore, the structural analysis of molecules belonging to the same olfactory family was shown to lead to the prediction of some receptors involved in their perception. These results constitute a basis for structure-odor relationships studies in the postgenomic era.

Les flavonoïdes, molécules naturelles possédant des propriétés antiradicalaires et antioxydantes, sont produits au cours de cascades enzymatiques impliquant plusieurs enzymes. Il a récemment été proposé que certaines de ces enzymes s'assembleraient pour former un complexe multienzymatique transitoire, appelé métabolon, ancré à la membrane cellulaire. Cette structure rendrait possible des phénomènes de transfert direct de métabolites d'un site actif à l'autre (substrate channeling), réduisant ainsi les temps de diffusion et minimisant les effets de solvatation/désolvatation du substrat. L'objectif de ce travail est de proposer un premier modèle de ce type de complexe, impliquant trois enzymes de la biosynthèse des flavonoïdes : la dihydroflavonol-4-réductase (DFR), la flavonoïd-3'-hydroxylase (F3’H) et la leucoanthocyanidine réductase (LAR). L'étude de ces complexes moléculaires requiert la mise en œuvre d'une méthodologie multi-échelles basée sur l’utilisation i) de méthodes hybrides QM/MM pour l'étude de la réactivité enzymatique, ii) de simulations de dynamique moléculaire à résolution atomique se déroulant sur des échelles de temps de l'ordre de la microseconde pour estimer des propriétés thermodynamiques et cinétiques, iii) de calculs de docking protéine-protéine en résolution gros grain. L'application des différents niveaux de théorie nous a permis d'établir un premier modèle de métabolon à trois enzymes en interaction avec une membrane cellulaire. / Flavonoids, some natural compounds exhibiting antioxidant properties, are synthesized along enzymatic cascades involving several enzymes. It has been recently proposed that some of these enzymes are involved in the formation of large transient macromolecular edifices, called metabolon, interacting with cellular membrane. Such molecular complexes should allow direct metabolites transfer from one active site to the other (substrate channeling phenomenon), minimizing diffusion time and solvation effects. The present work aims to establish a first model of a metabolon involving 3 enzymes of the flavonoid biosynthesis: the dihydroflavonol 4-reductase (DFR), the flavonoid 3'-hydroxylase (F3'H) and the leucoanthocyanidin reductase (LAR). The study of such large molecular system requires a multiscale approach based on i) hybrid QM/MM methods to decipher enzymatic mechanism, ii) molecular dynamic simulations in microsecond timescale to estimate thermodynamic and kinetic properties and iii) protein-protein docking at coarse-grained resolution. These different levels of theory allow us to establish a first model of a three-enzymes-metabolon in interaction with a model of cellular membrane.

Le TIMP-1, inhibiteur naturel des métalloprotéinases matricielles, exerce des effets pléïotropes indépendants de l'inhibition des MMPs et participe au développement de certains cancers et maladies neurodégénératives. Ces effets cytokiniques du TIMP-1 impliquent sa liaison à des récepteurs membranaires dont certains sont caractérisés, la glycoprotéine CD63/intégrine beta 1 et le complexe pro MMP-9/CD44. Cependant les acides aminés ou les domaines du TIMP-1 se liant à ces récepteurs ne sont pas identifiés. Les travaux réalisés au cours de cette thèse mettent en évidence un nouveau récepteur du TIMP-1, la protéine LRP-1. Dans les neurones corticaux murins, le TIMP-1 se fixe aux domaines DII et DIV de LRP-1, est endocyté et induit une réduction de la taille des neurites ainsi qu'une augmentation du volume des cônes de croissance. Afin de caractériser cette interaction, nous avons utilisé une approche originale de modélisation moléculaire associant les analyses de modes normaux et la dynamique moléculaire. Ces analyses in silico ont permis d'identifier un mouvement de pince entre les domaines N et C-terminaux du TIMP-1. Nous avons muté trois résidus (F12, K47 et W105) localisés dans une région essentielle d'un point vue énergétique à l'exécution de ce mouvement. Ces trois mutants n'ont pas d'effet sur la longueur du réseau neuritique et ne sont pas endocytés par LRP-1. En revanche, ils interagissent avec les 2 autres récepteurs (CD63 et proMMP-9) et reproduisent les effets du TIMP-1 sauvage. De plus, nous avons identifié une séquence de 6 acides aminés localisée dans le domaine extracellulaire I de CD63 et essentielle à la liaison avec le TIMP-1. L'ensemble de ces travaux a permis l'identification de régions impliquées dans l'interaction du TIMP-1 avec ses différents récepteurs et pourrait permettre le développement de nouveaux outils pharmacologiques ciblant les activités cytokiniques du TIMP-1. / TIMP-1, a natural inhibitor of matrix metalloproteinases, exerts pleiotropic effects independent of MMP inhibition and thus participates to the development of some cancers and neurodegenerative disorders. These cytokine-like activities require TIMP-1 binding to membrane receptors. Up to date two receptors, CD63/integrin beta 1 and proMMP-9/CD44, have been characterized. Nevertheless, TIMP-1 residues or regions binding these receptors remain unknown. In this work, we have identified the protein LRP-1 as a new receptor for TIMP 1. In mouse cortical neurons, TIMP-1 preferentially binds DII and DIV domains of LRP-1, is internalized via a LRP-1-dependent endocytosis, reduces neurite length and increases growth cone volume. To go deeper into TIMP-1/LRP-1 interaction, we used an original molecular modeling approach which combined normal mode analysis and molecular dynamic. These in silico studies allow us to point out a clamp movement between the N- and C-terminal domains of TIMP-1. Three residues localized in a region that seems essential for the movement have been mutated (F12, K47 and W105) and single mutants have been produced. These mutants do not reduce neurite outgrowth and are not internalized by LRP-1. In contrast, they interact with the two others receptors proMMP-9 and CD63 and induce associated biological effects. Furthermore, we have identified a sequence of six residues localized in the CD63 extracellular domain I and essential for TIMP 1 binding. The set of our data highlighted new regions of TIMP-1 interacting with its receptors and could lead to design novel therapeutic agents targeting the TIMP-1 cytokine like activities.

La consommation excessive et chronique de sucre est un facteur de risque pour l'apparition de pathologies telles que le diabète de type II ou l'obésité. Une des solutions pour répondre à cet enjeu majeur de santé publique, tout en conservant le plaisir de la saveur sucrée, consiste en l'utilisation d'édulcorants en substitution du sucre. Actuellement, un certain nombre d'édulcorants sur le marché présentent arrière-goût amer ou sont sujets à débat quant à leurs effets sur la santé. Un des objectifs de ces travaux de thèse consiste à proposer de nouveaux édulcorants grâce à des approches rationnelles in silico. Un modèle statistique a été établi sur la base des structures chimiques et a permis d'identifier de nouveaux édulcorants d'origine naturelle. Ensuite, la reconstruction par homologie du récepteur à la saveur sucrée et l'étude des sites de liaison apportent des indices, à l'échelle atomique, qui permettront d'identifier ou même de concevoir de nouveaux édulcorants. L'étude dynamique d'un récepteur de la même famille (Récepteur Couplé aux Protéines G (RCPG) de classe C) a permis d'émettre une hypothèse sur le mécanisme d'activation, phénomène important pour la compréhension de la conversion du signal chimique en signal électrique. / Sugar overconsumption is a risk factor for pathologies such as type II diabetes or obesity. Sweeteners consumption is used to overcome this public health issue. Indeed, they have low caloric value but still preserve the pleasure of sweet taste. Currently, number of sweeteners are commercially available, but they present a bitter aftertaste or there is a debate about their safety. One aim of this work was to propose new intense sweeteners using computational modeling strategies. Through a statistical approach to predict the sweetness based on the chemical structure of already known sweeteners, new natural compounds have been identified. Furthermore, the structural study of the homology model of the sweet taste receptor provides some clues to design new sweeteners. The molecular dynamic study of a class C G-protein coupled receptor gives the first molecular hypothesis of the activation process.

APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy.

Les leishmanioses sont un ensemble de maladies causées par un parasite du genre Leishmania. L'augmentation des résistances aux traitements actuellement disponibles conduit à la nécessité de développer de nouvelles molécules antileishmaniennes. La GDP-MP, une enzyme indispensable à la virulence du parasite a été choisie comme cible thérapeutique. Dans la première partie de ce travail, des modèles de GDP-MP de L. donovani et de H. sapiens ont été construits par modélisation moléculaire en utilisant la construction par homologie de séquence puis la relaxation par dynamique moléculaire. La conception d'inhibiteurs assistée par l'évaluation par amarrage moléculaire de ces molécules a conduit à l'identification de nombreuses molécules potentiellement inhibitrices de la GDP-MP. Parmi ces molécules identifiées, certaines sont des analogues de GDP-Mannose présentant différents motifs de remplacement pour le pont pyrophosphate ainsi que pour la guanosine. Une autre classe de composés sont les analogues de GDP portant un motif bisphosphoré. Une étude méthodologique portant sur l'obtention de conjugués 1,4-triazoles par CuAAC sur des analogues de guanosine a été conduite, et a mis en évidence, l'efficacité de l'utilisation de nanoparticules de cuivre (I) formées in situ par réduction du sulfate de cuivre en solution aqueuse par de l'hydrate d'hydrazine pour la synthèse de ces composés. Cette méthode, en association avec la chimie des H-phosphonates, a permis, l'accès à une librairie de dérivés de GDP-Mannose. De cette manière, des analogues de guanosine et de quinoléines diversement substituées ont été synthétisés. Les composés obtenus ont été évalués sur les GDP-MP recombinantes ainsi que sur cultures de parasites. Certains des composés bisphosphonates présentent à la fois une bonne inhibition de la GDP-MP et une bonne activité antiparasitaire. Les expériences futures de cristallographie des protéines permettront d'élucider le mode de fixation de ces composés et d'orienter les pharmacomodulations futures des inhibiteurs identifiés. / Leishmaniasis is a set of disease caused by a parasite of the genus Leishmania. The increased resistance to currently available treatments led to the need to develop new antileishmanial compounds. GDP-MP, an enzyme essential for the virulence of the parasite was chosen as a therapeutic target. In the first part of this work, GDP-MP models L. donovani and H. sapiens were constructed by molecular modeling using the method of sequence homology and molecular dynamics relaxation. The evaluation of inhibitors by molecular docking has led to the identification of many potential inhibitors of GDP-MP. Among these molecules identified, some are GDP-Mannose analogs having different substitution patterns for the pyrophosphate bridge and for guanosine. Another class of compounds is the analogues of GDP wearing bisphosphorus moiety. A methodological study for the synthesis of guanosine conjugated 1,4-triazoles analogs by CuAAC was conducted and showed the effectiveness of the use of copper nanoparticles (I), formed in situ by reduction of copper sulfate in aqueous solution of hydrazine hydrate, for the synthesis of these compounds. This method in combination with the H-phosphonate chemistry, has allowed access to a library of derivatives of GDP-Mannose. In this way, guanosine analogs, and variously substituted quinolines were synthesized. The obtained compounds were evaluated on the recombinant GDP-MP as well as parasite cultures. Some bisphosphonate compounds have both a good inhibition of the GDP-MP and good antiparasitic activity. Future experiments of protein crystallography will elucidate the mode of binding of these compounds and will determine future pharmacomodulations on the identified inhibitors.

Insulin receptor is a multi-domain signalling protein acting as a dimer. It comprises an extracellular ectodomain, a transmembrane domain and intracellular tyrosine kinase domain. Upon insulin binding, conformational changes in insulin as well as in insulin receptor occur and trigger the signaling cascade via the kinase domain. Abnormalities in insulin and insulin receptor function cause diabetes mellitus, a widespread disorder which can be consequence of genetic factors as well as lifestyle and is manifested by increased level of blood glucose. A common treatment of diabetes mellitus is via insulin analogues with different molecular properties. Insulin/insulin receptor interactions in the binding pocket are divided into two groups, so-called "site1" and "site2". The molecular details of the interactions in site1 are well known, while site2 residues are still not completely elucidated. It is important to shed light on the binding properties of insulin and insulin receptor, especially site2 interactions, because it could contribute to improved design of new insulin analogues. In this work, we used the very recent breakthroughs in the structural biology of insulin receptor to study the interactions by computational chemistry methods. It was thus possible to assess the noncovalent interactions and...

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Nanoscale materials have become an active area of research due to the enhanced mechanical properties of the nanomaterials in comparison to their respective bulk materials. The effect that the size and shape of a nanomaterial has on its mechanical properties is important to understand if these materials are to be used in engineering applications. This thesis presents the results of molecular dynamics (MD) simulations on copper, gold, nickel, palladium, platinum, and silver nanowires of three cross-sectional shapes and four diameters. The cross-sectional shapes investigated were square, circular, and octagonal while the diameters varied from one to eight nanometers. Due to a high surface area to volume ratio, nanowires do not have the same atomic spacing as bulk materials. To account for this difference, prior to tensile loading, a minimization procedure was applied to find the equilibrium strain for each structure size and shape. Through visualization of the atomic energy before and after minimization, it was found that there are more than two energetically distinct areas within the nanowires. In addition, a correlation between the anisotropy of a material and its equilibrium strain was found. The wires were then subjected to a uniaxial tensile load in the [100] direction at a strain rate of 108 s-1 with a simulation temperature of 300 K. The embedded-atom method (EAM) was employed using the Foiles potential to simulate the stretching of the wires. The wires were stretched to failure, and the corresponding stress-strain curves were produced. From these curves, mechanical properties including the elastic modulus, yield stress and strain, and ultimate strain were calculated. In addition to the MD approach, an energy method was applied to calculate the elastic modulus of each nanowire through exponential fitting of an energy function. Both methods used to calculate Young’s modulus qualitatively gave similar results indicating that as diameter decreases, Young’s modulus decreases. The MD simulations were also visualized to investigate the deformation and yield behavior of each nanowire. Through the visualization, most nanowires were found to yield and fail through partial dislocation nucleation and propagation leading to {111} slip. However, the 5 nm diameter octagonal platinum nanowire was found to yield through reconstruction of the {011} surfaces into the more energetically favorable {021} surfaces.