The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of MBD2 and MBD3. This study provides information regarding previously unidentified MBD3 binding properties and potential biological function. In chapter three, we show that sponges demonstrate a MBD2-mediated capacity for binding methylated DNA sites, recruit NuRD components in vitro, and knockdown of MBD2 in the freshwater desmosponge, Ephydatia muelleri, promotes an abnormal growth phenotype.

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

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La capacité du virus de la grippe aviaire à traverser la barrière des espèces et à devenir fortement pathogène chez les mammifères est un problème majeur de santé publique. Chez les oiseaux, la réplication a lieu dans l'intestin, à 4C, tandis que chez les humains elle a lieu dans l'appareil respiratoire, plus froid, à 33C. Il a été montré que l'adaptation à la température du virus de la grippe a lieu par de nombreuses mutations de la polymérase virale, notamment dans le domaine 627-NLS situé en C-terminal de la protéine PB2. Ce domaine est impliqué dans l'adaptation à l'hôte et interagit avec la protéine de l'hôte, importine alpha, étant donc indispensable pour l'entrée de la polymérase virale dans le noyau de la cellule [Tarendeau et al., 2008]. Les structures cristallographiques du 627-NLS et du complexe importine alpha/NLS existent. Cependant, lors de la superposition de ces structures via leur domaine NLS commun, un important choc stérique entre le domaine 627 et l'importine alpha devient évident. Ceci indique qu'une autre conformation du 627-NLS est requise pour l'interaction avec l'importine alpha [Boivin and Hart, 2011]. Dans cette étude, nous avons examiné les bases moléculaires de l'adaptation inter-espèces du virus à travers l'étude de la structure et de la dynamique du 627-NLS aviaire et humain. Nous avons identifié deux conformations du 627-NLS en échange lent (10-100 s-1), correspondant apparemment à une conformation ouverte et une conformation fermée des deux domaines. Nous proposons que la conformation ouverte du 627-NLS est la seule conformation compatible avec l'interaction avec l'importine alpha, et que l'équilibre entre conformation ouverte et fermée pourrait jouer le rôle de thermostat moléculaire, contrôlant l'efficacité de la réplication virale chez différents hôtes. La cinétique et la dynamique de ce comportement conformationnel important ainsi que de l'interaction entre le 627-NLS et l'importine alpha ont été caractérisées par résonance magnétique nucléaire (déplacements chimique, augmentation paramagnétique de la relaxation, relaxation de spin, transfert de saturation par l'échange chimique), combinée à la diffusion des rayons X et des neutrons aux petits angles ainsi qu'au transfert d'énergie par résonance de type Förster. Aussi, nous avons déterminé les affinités d'une série de mutants évolutifs du 627-NLS pour l'importine alpha et du 627-NLS aviaire ou humain pour différents isoformes de l'importine alpha, montrant que les affinités observées sont cohérentes avec les préférences d'interactions vues in vivo. / The ability of avian influenza viruses to cross the species barrier and become dangerously pathogenic to mammalian hosts represents a major threat for human health. In birds the viral replication is carried out in the intestine at 40°C, while in humans it occurs in the cooler respiratory tract at 33°C. It has been shown that temperature adaption of the influenza virus occurs through numerous mutations in the viral polymerase, in particular in the C-terminal domain 627-NLS of the PB2 protein. This domain has already been shown to participate in host adaptation and is involved in importin alpha binding and therefore is required for entry of the viral polymerase into the nucleus [Tarendeau et al., 2008]. Crystallographic structures are available for 627-NLS and the complex importin alpha/NLS, however, a steric clash between importin alpha and the 627 domain becomes apparent when superimposing the NLS domain of the two structures, indicating that another conformation of 627-NLS is required for binding to importin alpha [Boivin and Hart, 2011]. Here we investigate the molecular basis of inter-species adaptation by studying the structure and dynamics of human and avian 627-NLS. We have identified two conformations of 627-NLS in slow exchange (10-100 s-1), corresponding to an apparently open and closed conformation of the two domains. We show that the equilibrium between closed and open conformations is strongly temperature dependent. We propose that the open conformation of 627-NLS is the only conformation compatible with binding to importin alpha and that the equilibrium between closed and open conformations may play a role as a molecular thermostat, controlling the efficiency of viral replication in the different species. The kinetics and domain dynamics of this important conformational behaviour and of the interaction between 627-NLS and importin alpha have been characterized using nuclear magnetic resonance chemical shifts, paramagnetic relaxation enhancement, spin relaxation and chemical exchange saturation transfer, in combination with X-ray and neutron small angle scattering and Förster resonance energy transfer. Also, we have determined the affinities of various evolutionnary mutants of 627-NLS to importin alpha and of avian and human 627-NLS to different isoforms of importin alpha, showing that the observed affinities are coherent with the preferred interactions seen in vivo.

Le virus respiratoire syncytial humain (hRSV) est le principal agent pathogène responsable des bronchiolites. Le complexe ARN polymérase (RdRp), du hRSV, nécessaire à la réplication de son génome, est composé a minima de la sous-unité catalytique (L), de son principal cofacteur qu’est la phosphoprotéine (P) et de la nucléoprotéine (N) qui assure l’encapsidation du génome viral. Le cœur de mon projet doctoral a été l’étude dynamique et structurale de domaines des protéines N et P du hRSV ainsi que leurs interactions avec certaines protéines cellulaires principalement par résonance magnétique nucléaire.Dans un premier temps j’ai étudié une potentielle interaction entre 2 domaines appartenant à la protéine N et à la protéine cellulaire Tax1BP1 impliquée notamment dans la régulation de l’autophagie. Ensuite, j’ai entrepris une étude structurale et dynamique de hRSV-P isolée notamment dans le but de déterminer des contacts transitoires au sein de la protéine et d’obtenir la structure tridimensionnelle du domaine d’oligomérisation de P. Enfin, j'ai participé à la caractérisation de l’interaction entre la protéine hRSV-P et le cofacteur de transcription du hRSV hRSV-M2-1, puis entre hRSV P et la phosphatase cellulaire PP1α, afin d’en cartographier les régions de contacts. / Human respiratory syncytial virus (hRSV) is the main pathogen responsible for bronchiolitis. The RNA polymerase complex (RdRp) of hRSV, necessary for the replication of its genome, is composed at least of the catalytic subunit (L), its main cofactor phosphoprotein (P) and nucleoprotein (N), which encapsidates the viral genome. At the heart of my doctoral project was the dynamic and structural study of domains of the proteins N and P of the hRSV as well as of their interactions with several cellular proteins, mainly by nuclear magnetic resonance.Firstly, I studied a potential interaction between 2 domains belonging to the N protein and to the Tax1BP1 cellular protein involved notably in regulation of autophagy. Secondly, I undertook a structural and dynamic study of isolated hRSV-P in order to determine transient contacts within the protein and to obtain the three-dimensional structure of the P oligomerization domain. Last, I participated in the characterization of the interaction between the hRSV-P protein and the hRSV transcription cofactor hRSV-M2-1, and between hRSV P and the cellular phosphatase PP1α to map the contact regions.

For many enzymes to function correctly they must have the freedom to display a level of dynamics or communication during their catalytic cycle. The effects that protein dynamics and communication can have are wide ranging, from changes in substrate specificity or product profiles, to speed of reaction or switching activity on or off. This project investigates the protein dynamics and communication in two separate systems, a non-ribosomal peptide synthetase (NRPS), and a protein arginine methyltransferase (PRMT). PRMT1, the enzyme responsible for 80% of arginine methylation in humans, has been implicated in a variety of disease states when functioning incorrectly. For this reason, much focus has been placed on better understanding how PRMT1 determines which products it creates and at what times. This project aims to shed light on how dynamics and communication within PRMT1 dictate its activity. We have to this point developed a protocol for creating and purifying a linked PRMT1 construct which will enable us to conduct the necessary experiments capable of answering our larger questions about the PRMT1 catalytic mechanism. Our collaborators in the Zhan lab discovered the presence of a methyltransferase (Mt) in the two NRPS systems they study, which produce two different and medically relevant compounds, bassianolide and beauvericin. The Hevel lab is well suited to study methyltransferases and so were asked to help evaluate the role of these Mt domains and how they affect the production of the relevant natural products. Achieving a more complete understanding of these systems will move us closer toward the “holy grail” of being able to manipulate and harness NRPS systems for the engineering of novel medically relevant compounds. This project has found that the Mt domain substrate specificity is affected by the surrounding protein domains, or even small portions of them.

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