Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.

[Truncated abstract] The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to changes in extracellular calcium and plays an integral role in calcium homeostasis. Later studies revealed that the CaR was stimulated by not just calcium, but a diverse range of stimuli and that activation of the receptor regulated a host of different biological processes. The CaR is linked to these cellular responses via the various signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have identified a number of accessory proteins that, through their interaction with the intracellular tail of the CaR, are able to regulate important functional aspects of the receptor, including its signalling and degradation. We hypothesised that many more proteins that bind to the CaR-tail await identification, especially since most of the previous studies used the yeast two-hybrid system to screen cDNA libraries generated from tissues that are important to whole body calcium homeostasis, such as the parathyroid gland and kidney. In order to identify novel binding partners of the CaR, which may affect its function, particularly in biological processes that might be unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular tail of the human CaR as bait. This screen revealed a large number of

The last few decades have witnessed an upsurge in the availability of large-scale data on genomes and genome-scale information. The development of methods to understand the trends and patterns from large scale data promised potentially to unravel the mechanisms responsible for the enormous diversity observed in biological systems. Of the many mechanisms adopted, protein-protein interactions represent one of the commonly adopted mechanisms to achieve functional diversity using a limited genetic repertoire. Protein-proteins interactions bring about several fundamental cellular processes and also modulate regulation at the cellular level. Different types of protein-protein interactions have evolved to carry out myriad functions in a cell. Mainly, interactions can be permanent or transient in nature, depending on the duration of interaction. In terms of affinity ,they are classified as obligate or non-obligate interactions. Structural studies on the various kinds of complexes have enabled the identification of the distinctive features characterizing the different types of complexes. Further, identifying the mechanisms involved in the evolution of protein-protein interactions are important in understanding the forces involved in their maintenance. Such studies also provide clues for the development of methods to predict protein-protein interactions from the large repertoire of sequence and structural data available. In spite of significant understanding of various aspects of protein-protein interactions, several questions still remain unanswered. The work embodied in this thesis studies two main aspects of protein-protein interactions for various types of complexes: structural and evolutionary features. The first part of the thesis(comprising of chapters 2,3,4 and 5) involves structural studies on the following features of protein-protein interactions: structural change, flexibility, symmetry, and residue conservation. The second part of the thesis(comprising of chapters 6,7,8 and 9) involves study of evolutionary aspects of protein-protein interactions, based on both large-scale data as well as case studies. Chapter1 provides a background and literature survey of the area of protein-protein interactions. The different classification schemes commonly used for describing the various protein-protein interactions are outlined. The key small-scale and large-scale experimental methods used for the identification of protein-protein interactions are described along with the details of the databases storing such experimentally derived information. Further, a comprehensive account of structural and evolutionary studies performed so far using the available data is provided. The computational(prediction)methods developed to address various aspects of protein-protein interactions are also outlined. In addition, the importance of protein-protein interactions in the context of diseases and the development of methods used to inhibit these interactions are discussed. Finally, the efforts towards design of protein-protein interactions based on the understanding of the principles governing their formation are outlined. Chapter 2 and chapter 3 describe different aspects of transient protein-protein interactions, which form an important subset of interactions, and are mainly involved in the regulation of Cellular processes. In chapter 2, the structural changes occurring upon complex formation are described. In chapter 3, roles of interface residues in the unbound form are described. In chapter 2, the nature, extent and location of structural changes upon binding is analyzed using a non-redundant and curated dataset of 77 structures of protein-protein complexes available in both bound and unbound forms. Structural change has been captured using two metrics: protein blocks and root mean square deviation of Cα positions. The relevance of the structural changes observed in protein-protein complexes is established by comparison with a control dataset of proteins not bound to any small or macromolecule. Results indicate that the observed changes are much larger than those observed due to random fluctuations. Given this background, the following observations were made on the nature, extent and location of structural changes in protein-protein complexes.(i) The nature of structural changes occurring at the interface is largely conformational with few rigid-body movements.(ii)The interfaces in the dataset are segregated into three types based on the extent of structural changes at the interface. A significant fraction of the interfaces are ‘pre-made’(almost in variant interface) or‘ induced-fit’(interface with large structural changes), while the rest are interfaces with moderate structural changes(‘others’). Analysis of structural changes using protein blocks reveals that pre-made interfaces are not completely invariant and are characterized by conformational changes of small magnitude. Pre-made interfaces are also observed to bind preferentially to‘ induced fit ’or‘ other’ interfaces. These observations implicate that non-obligate interactions possess in-built regulatory mechanisms in terms of conformational features to control the timely association-dissociation of transiently interacting proteins. (iii) Interestingly, significant structural changes away from the interface were observed in almost one-half of the complexes in the dataset. The analysis of these changes forms a major focus of this chapter. Crystallographic temperature factors, crystal packing, and normal mode analysis of these regions were studied to analyze the structural changes in these regions. Normal mode analysis along with literature survey indicates that most of the structural changes observed in non-interacting surface regions may be functionally relevant, with many of them corresponding to allosteric transitions. The majority of these changes occur in signaling proteins. The chapter summarizes that these observations suggest a much higher prevalence of allostery caused due to protein binding than appreciated before. The data set generated in this chapter can serve as a starting point to uncover potentially new allosteric modulators in signaling systems. In chapter 3, the question‘ Do residues at the interface of transient protein-protein interactions have any role in the unbound form?’ has been investigated. A high resolution, non-redundant and functionally diverse dataset of 67 proteins with known structures available both in the form of protein-protein complex and unbound forms has been used. Significantly low B-factors at the core of the interface in the unbound form are observed in these structures, indicating high rigidity. Many of these residues also show B-factors comparable to those of buried residues in a protein, which formed the basis for classifying interface residues as ‘rigid’ and‘ non-rigid’. These two types of residues have differential contribution towards the energetics of complex formation. It is also observed that rigid interfacial residues are conserved better in evolution than non-rigid interfacial residues. Their stronger selection is highlighted by substantial conservation of microenvironment (rigidness), sequence(amino acid identity/similarity) and structure(specific side-chain orientation) in homologous proteins. These observations coupled with the absence of any specific type of amino acid to occur preferentially at a rigid site indicates that rigidness is a property of the topological location of the residue at the interface and not the type of the amino acid present at that site. Analysis of the energetic parameters of these residues indicates that the y contribute significantly to the molecular recognition process by reducing the entropic cost on complexation by virtue of their pre-ordered conformation. This chapter also explores the contribution of interface residues towards the stability of the self-protein vis-à-vis that of the complex. It was seen that most interface residues contribute towards stabilizing the bound form. Interestingly, some of the interfacial residues predominantly stabilize the self-protein(the protein in which they are situated) and have a negligible contribution towards stabilization of the bound form. Thus, though these residues are located in the protein-protein interface their main role seems to be in the stabilization of the self-protein both in the unbound and bound forms. These residues are classified as Self-protein Stabilizing Residues(SSR -6.93%) and the rest as Neutrally Stabilizing Residues(NR -42.60%) and Complex Stabilizing Residues(CSR -50.46%). In addition, it was noted that the proportion of rigid residues is more in SSR(73.33%) than in NR(58.13%) and CSR(48.90%)sites. Apart from the favorable energetic contribution by rigid residues to the free energy of the unbound form than non-rigid residues, their predominance in SSRs suggests that rigid residues play an important role in the stabilization of the unbound form of the protein.The analyses performed in this chapter suggest that not all the protein-protein interfacial residues have the major role of stabilizing the complex; some of these residues seem to have more significant role in the unbound form than the bound form. Chapter4 provides a discussion on the prevalence and relevance of a symmetry in homodimeric proteins. One of the main features characterizing homodimers is the symmetric arrangement of subunits in the three-dimensional structures.Typically, asymmetric arrangements of subunits are associated with disease states; however, they are also observed in normal homodimers performing specialized functions. Two measures to quantify structural asymmetry in homodimers (global asymmetry and interface asymmetry)have been used on an on-redundant dataset of 223 biologically relevant homodimers. The survey for globally asymmetric homodimers in the dataset indicates that they are very rare(n=11).The chapter discusses cases where a globally asymmetric arrangement of homodimeric proteins has been utilized by the nature to perform certain specialized functions, such as linking of a dimeric system with a monomeric system(half-of-sites reactivity) and the transmission of signals emanating from asymmetric DNA repeats. Analysis of the 3-D structures of homologues reveals that there is no clear conservation of asymmetry. Specifically, the function of the homologous protein appears to dictate the pattern of structural organization. This chapter also describes the structural and evolutionary analyses of the 11 globally asymmetric complexes, which suggest possible mechanisms adopted by nature for preventing infinite array formation. The postulated mechanisms are:(i) In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used.(ii) In case of homodimers associating via partially topologically equivalent surfaces, steric hindrance serves as the preventive mechanism of infinite array. Since most of the biologically relevant homodimers exhibit gross structural symmetry, this chapter explores further the extent of interface asymmetry in symmetric homodimers. It was observed that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Further, binding of small ligands at the interface does not cause any significant variation in interface asymmetry.The chapter provides new insights regarding accommodation of structural asymmetry in homodimers. Chapter 5 describes the ability of residue conservation of interface residues vis-à-vis surface residues near interface residues to identify fitting errors caused due to mis-orientation in cryo-electron microscopy maps. Cryo-electron microscopy is the most popular technique for solving structures of large assemblies in physiological conditions. However, the structures are usually solved at low resolution and atomic resolution is desired to get insights at the molecular-level. Although several methods have been developed for the fitting of atomic structures or models in to low-resolution cryo-electron microscopic maps, inaccurate fitting is observed in several cases. Using a non-redundant and high-resolution dataset of 125 permanent interactions and 95 transient interactions, it was observed that interface residues are significantly conserved better than residues near to the interface. The chapter describes the ability of this differential conservation to identify probable mis-fittings in cryo-EM maps for three case-studies: ribosomal complex from Escherichia coli, transferring-transferrin receptor complex from Homosapiens, and glutamate synthase complex from Azospirillum brasilense. For these cases, the use of conservation information resulted in the identification of a few residues in the vicinity of the interface with significantly higher conservation, implying their probable occurrence in the interface. These findings were verified against the high-resolution structures for two of these complexes (ribosomal assembly and transferring-transferrin receptor complex).These analyses suggest that residue conservation information can be useful in the fitting process to arrive at the fitted structure with an improved accuracy. Further, the discriminative power of the simplistic measure of residue conservation coupled with residue surface accessibility in identifying interacting residues on protein structures is also analyzed in this chapter. Testing on a set of signaling and scaffolding molecules indicates that this simplistic measure can identify interface residues in protein structures, indicating that conservation contains a distinct, although weak, signal for functional regions. Chapters 6 to 9 discuss studies involving evolutionary aspects of protein-protein interactions. Chapter 6 describes the usage of phylogenetic tree construction using maximum likelihood method to understand the origin of the signal captured by the mirror tree approach. Mirror tree is one of the most popular approaches for identifying interacting proteins based on co-evolution. This method uses the similarity in phylogenetic trees as an indicator of protein-protein interaction. The origin of the evolutionary signal detected by the mirror tree method is a subject of some controversy. Two broad hypotheses have been postulated in the literature to explain the origin of the signal(i)site-specific co-evolution alone and(ii)correlation induced by external factors with only minor, if any, contribution from site-specific co-evolution. In the typical mirror tree protocol, inferences from phylogenetic tree are optional and only genetic distances are analysed. Even if the tree is constructed, usually the Neighbor-Joining approach is used. However ,with Neighbor-Joining method the inferred tree topology and genetic distances are directly linked. With maximum likelihood the tree topology is not derived directly from the genetic distances and therefore the contributions of the signals arising from tree topology and genetic distances can be studied separately. Tree topologies can be considered to serve as indicators of compensatory substitutions(implicated in site-specific co-evolution)as well as shared evolutionary history. Genetic distances correspond to evolutionary rates(implicated in correlation induced by external factors).Using this method, phylogenetic trees for a range of datasets of interacting and non-interacting proteins corresponding to yeast(S.cerevisiae) have been derived. The analysis performed in this chapter reveals no strong correlation between phylogenetic tree topologies, and significant correlation between genetic distance matrices for interacting proteins. The chapter discusses the implications of these findings and attempts to understand the origin of the signal captured by mirror tree protocol using the following points.(i) The near lack of correlation in tree topologies is not surprising since compensatory substitutions accounts for only a minority of the sites in a protein.(ii) The influence of shared evolutionary history has also been tested in the chapter by comparison of tree topologies of interacting proteins and non-interacting with 18S rRNA tree. Tree topologies of both interacting and non-interacting proteins do not mirror the 18S rRNA tree, ruling out shared evolutionary history as the signal of correlated evolution.(iii) By contrast, the significant correlation observed between branch lengths(genetic distances) of interacting proteins in all the variant datasets demonstrates correlation between evolutionary rates, independent of evolutionary divergence. In summary, the chapter concludes by providing support for the theory of correlation induced by external factors with only minor contribution from site-specific co-evolution. Chapter 7 explores the homology based transfer of interactions by quantifying the extent of retention/variation of interaction partnerships amongst a set of homologous proteins related at SCOP family level(which indicates clear evolutionary relationship).A large dataset of domain-domain interacting pairs(n=20,254)culled from SCOP1.73 was used for this analysis. Study involving this dataset shows that in around~80% of the cases, interacting partners are completely retained(evaluated as proteins belonging to the same SCOP family).If‘common’ partnership is evaluated at the level of SCOP folds, which are known to be structurally similaral though not necessarily evolutionarily related, the percentage of homologous domains with complete retention of partnership increases only by~5%. This indicates that only the presence of a common structural scaffold is not a sufficient feature for interaction. Further, the chapter also describes the retention/variation in partnerships analyzed as a function of sequence divergence between the homologous proteins. It is observed that there is a higher tendency to vary interacting partners as the evolutionary divergence between the homologues increases. In spite of this, interaction partnerships appear to be retained for homologous domains irrespective of their sequence divergence if the function mandates the presence of the interaction. However, all these observations could be influenced by the incomplete nature of information on the interactions available in the structural space. This problem has been addressed in this chapter by studying variation in interaction partnerships for Saccharomyces cerevisiae proteins. Yeast was chosen since it is extensively studied and interactions are available for~87% of proteins yielding a comprehensive list of interactions. To study this aspect, the SCOP dataset of interacting proteins(which represents a generic dataset) was compared with interactions of homologous proteins from yeast. The dataset of interacting proteins for yeast collated from all sources and documented in BIOGRID v50 was used. In this analysis, the proportion of homologous domains showing complete retention of interacting partners was only ~12%. This observation is the reverse of the trend observed for the dataset of homologous SCOP domains. Further analysis of homologous pairs of yeast-SCOP domains, containing only those pairs whose interacting protein families are found both in yeast and SCOP dataset, was performed to ascertain the extent of contribution of organism-specific proteins to the variation in interaction partnership for homologous domains. The proportion of homologous domains showing complete retention of interaction partners increases to~50% for these cases. These observations indicate that organism-specific proteins contribute significantly to the variation of interaction partnerships in homologous proteins. The next two chapters(8 and 9) discuss two contrasting scenarios of interaction partnerships. Chapter 8 describes the study of two protein families showing variation in interaction partnerships/interface structure and analyzes the drift in protein-protein interaction surfaces in each of the cases. The analysis in this chapter is facilitated by the large number of sequences available for the case studies. The first case study involves members of the glutamine amido transferase (GAT) superfamily of enzymes. Three remote homologues in this superfamily could also be related by sequence: intracellular protease(DJ-1/PfpIfamily),C-terminal domain of the small subunit of carbomoyl phosphate synthetase (ClassI glutamine amidotransferase-like family), and C-terminal domain of catalase (Catalase ,C-terminal domain family).In two cases, it is seen that domain recruitment influences the interacting surface(catalase, carbamoyl phosphate synthetase). The tethered domains, which are involved in interaction with the GAT domain, are from different SCOP folds, indicating that partnerships are not retained at extreme divergence. However, members of the DJ-1/PfpIfamily form homodimers with differing quaternary structures i.e. different orientations of the dimers. Four members have been studied in detail in this chapter (intracellular protease–two distinct interfaces–forming hexamer, stress-induced protein -dimer, DJ-1protein -dimer, sigma cross-reacting protein -dimer). Since the members are sequentially less divergent(as they are within the same family), it is possible to trace the drift in interfaces among these members based on the multiple sequence alignments of members with the differing quaternary structures and the sequences bridging them. The second case study involves analysis of the family of legume lectins, which corresponds to another set of proteins exhibiting differing quaternary structures for remarkably well conserved tertiary structures and sequences. Analysis of variations in protein-protein interaction surfaces when they show only slight differences between homologous members indicates that the drift is gradual, as seen when tracing the dynamics of DJ-1 family members and legume lectin family members. There exist sequences containing many different intermediate combinations of the interacting residues involved in both the sets of proteins. Comparisons of homologues where an entire interface seems to be lost show a different trend(intracellular protease and DJ-1).The most prominent interacting residues show an abrupt shift between the two different subfamilies. However, inspection of the other interacting residues reveals that there is a gradual change occurring generally, although a drastic change in the important(although quantitatively smaller) residues would have led to loss of interface. In summary, analysis of the evolutionary dynamics of the consensus interface residues of different quaternary structure types of DJ-1/PfpI family of enzymes and legume lectins shows that nature employs only the most important mutations to Prevent a specific interface and form a new interface and the rest of the positions drift and accumulate changes in the course of evolution. Chapter 9 describes the opposite scenario i.e. conservation of an interface even at high sequence divergence, using the RNA polymerase assembly as a case study. The multi-molecular assembly consists of four core subunits–alpha (I and II), beta, betaprime, and omega. These four subunits are common to RNA polymerase complexes of eubacteria, eukaryota and archaea. The sigma subunit aids in initiation of transcription in eubacteria (cor eenzyme +sigma = holoenzyme). Remarkably, prokaryotic and eukaryotic structures exhibit high degree of structural similarity, although their sequence similarity is low(19-28% sequence identity).However, this is expected as the obligatory interaction between the various subunits is essential to successfully carry out transcription. This chapter investigates the structural accommodation of diverse sequences at the interface of RNA polymerase machinery of eubacteria, using sequence analysis and homology modelling. Analysis of domain composition and order of domains for the core subunits of the RNA polymerase assembly in>85 eubacterial species indicates complete conservation. However, conservation analysis of the various core subunits indicates that the interface residues are more divergent for alpha and omega subunits. Although beta and beta prime are generally well-conserved, the residues involved in interaction with the divergent subunits(i.e.alpha, omega) are not conserved. Insertions/deletions are also observed near the interacting surfaces even in the cases of most conserved subunits(beta and betaprime). The chapter describes the homology modeling of three divergent RNApolymerase complexes from Helicobacter pylori, Mycoplasma pulmonis and Onion yellows phytoplasma, highlighting that insertions/deletions can be accommodated near the interface as they generally occur at the periphery. The development of a generalized matrix capturing preferences of interface environment is documented, along with results comparing the similarity of the modeled interfaces to that of the template interface. It is observed that the modeled interfaces are physico-chemically similar to that of the template interfaces in Thermus thermophilus, indicating that nature accommodates substantial substitutions and insertions/deletions at and near the interface in order to retain the structure of the obligate complex, which is in dispensable for the process of transcription. The main conclusions of the entire thesis work are summarized in chapter10, which also places the work in the context of the field of protein-protein interactions. The new insights obtained for transient interactions and homodimers from structural studies are highlighted. The application of evolutionary conservation to improve fitting of atomic structures in cryo-electron microscopic maps is discussed. The understanding gained from study of different evolutionary aspects of protein-protein interactions, ranging from correlated evolution to evolutionary dynamics of variations in interactions is also highlighted. Appendix 1 of this thesis describes the homology modeling of the hexameric form of AAA ATPase domain of spastin along with associated structural analysis.

L’identification de molécules organiques capables de moduler des interactions protéine-protéine (PPIs) est longtemps restée un domaine peu exploité par la recherche pharmaceutique privée comme académique. Cependant, le développement de méthodologies innovantes pour l’étude des PPIs et la validation récente de ce type d’inhibiteurs dans des essais précliniques, démontrent que les PPIs constituent une nouvelle source de cibles importantes. Les composés capables de moduler ces interactions représentent une nouvelle classe d’outils prometteurs, tant en recherche fondamentale qu’en thérapeutique. Elles peuvent aider à différencier les multiples fonctions portées par une même protéine, à replacer la protéine dans une cascade de réactions, ainsi qu’à disséquer et reconstituer des réseaux de signalisations protéiques. Ces molécules permettront également de faire émerger de nouvelles familles d’agents pharmacologiques actifs dans diverses pathologies.Mon travail de thèse s'est projeté dans l'avenir de la recherche biomédicale, en ciblant les interactions protéine-protéine. J’ai pu durant mon doctorat mettre en œuvre plusieurs méthodologies pour étudier et caractériser des interactions protéiques afin de développer des inhibiteurs de ces interactions. J’ai ainsi pu travailler sur l’optimisation d’un composé inhibiteur de l’interaction de la protéine virale Nef VIH-1 avec les domaines SH3 des Src kinases, le composé DLC27. J’ai également pu mettre en évidence la pertinence biologique de ce composé, qui cible un mode d’interaction unique, ou très rare, au niveau cellulaire en étudiant l’interaction avec les domaines SH3 de deux protéines, ALIX (ALG2-Interacting Protein X) et la sous-unité p85 de la PI3K (phosphatidylinositol 3-kinase).J’ai également pu caractériser la surface et le mode d’interaction de protéines virales impliquées dans le complexe de réplication du virus du SRAS (Syndrome Respiratoire Aigu Sévère). Cette étude tend à montrer que la protéine virale nsp10 agit comme une plateforme de reconnaissance pour ses partenaires, les protéines virales nsp14 et nsp16. Ces interactions permettent l’activation ou l’augmentation des activités respectives de nsp16 et nsp14 et jouent un rôle au niveau de la réplication virale. Suite à l’identification d’un ‘point chaud’ d’interaction, le résidu Tyr96 à la surface de nsp10, nous avons mis en évidence la première famille de molécules inhibitrices du complexe nsp10-nsp14 en couplant des méthodes informatiques (in silico) à des criblages expérimentaux. Ces molécules pourraient être utilisées comme antiviraux ou servir d’outils pour la recherche, en permettant par exemple de mieux comprendre et d’élucider les mécanismes moléculaires impliqués dans la réplication du virus du SRAS et des coronavirus en général. / Protein-protein interactions (PPIs) participate in and regulate almost all essential cellular functions. As a consequence, they are frequently involved in various pathologies (going from cancer development to viral replication and host cell infection) but their study remains a challenge.Thus understanding those interactions as well as finding small drug candidates able to modulate them, a field of research not currently fully developed, appear as the future of the healthcare industry.In this context, I chose to learn different techniques to study PPIs that are usually employed in academic (IMR laboratory, CNRS, France) or corporate environments (Genentech, USA). Moreover, I also worked on the development of small organic inhibitors of PPIs coupling in silico methodologies (chemo-informatics, Drug Design) to biological and structural validations.During my PhD, I could manage and work on different projects involving the study of PPIs involved in cancer signaling pathways as well as the development of potent antiviral drugs targeting the HIV and SARS viruses.My organizational, personal and scientific skills as well as the practical experience I developed on various techniques (from cell biology to biophysics, structural biochemistry and Drug Design), make me feel confident on the management of PPIs drug discovery projects.I am thus able to efficiently work on, and manage, the study of protein-protein interactions in various pathologies as well as the development of potent PPIs inhibitors, that will be a major breakthrough for Biotech/Pharma companies in the coming years.

Le nombre considérable d'interactions protéine-protéine (PPIs) existant au sein d'un organisme, ainsi que leur implication cruciale dans la vie cellulaire et dans de nombreuses pathologies, font des PPIs un immense réservoir de cibles potentielles pour la recherche de médicaments. Les PPIs sont aujourd'hui sur le devant de la scène grâce au développement de méthodologies innovantes et la validation récente de molécules chimiques modulant ces interactions dans des essais précliniques.L'étude des modulateurs d'interactions protéine-protéine (PPIM), a des implications tant dans la recherche fondamentale que thérapeutique. Les PPIMs peuvent aider à la compréhension des réseaux d'interactions. Elles permettront également de faire émerger de nouvelles familles d'agents thérapeutiques actifs dans diverses pathologies.Mon travail de thèse a principalement porté sur deux aspects de l'étude de l'inhibition des PPIs. D'une part, l'étude de l'implication des divers paramètres physicochimiques gouvernant une PPI dans sa capacité à être modulée (étude dite de la « druggabilité »), m'a amené à participer à la création d'une base de données structurale des interactions protéine-protéine : 2P2IDB (http://2p2idb.cnrs-mrs.fr/). D'autre part, j'ai contribué à l analyse de l'espace chimique des molécules présentes dans la base de données 2P2IDB. Nous avons défini la « Rule Of 4 » comme ligne de conduite pour caractériser ces molécules. Nous avons de plus utilisé le SVM afin de créer un protocole innovant (2P2IHUNTER) qui nous a permis de filtrer de grandes collections de composés afin de créer des chimiothèques dédiées aux PPIs. / The number of protein-protein interactions (PPIs) existing in an organism, and their crucial implication in cellular life and in many pathologies, demonstrates the importance of PPIs as a large reservoir of potential targets for medicinal research. Neglected for a long time by both pharmaceutical companies and academic laboratories because they were historically classified as difficult targets, PPIs are now getting into the groove due to the development of innovative methodologies and the growing number of small molecule compounds modulating these interactions.The study of PPI modulators has implications in both fundamental and therapeutics research. On the one hand, PPI modulators can be used in basic research to decipher the role of PPIs in biological networks. On the other hand, they represent a valuable source of new families of therapeutic agents in pathologic processes.In the first part of my PhD, I contributed to the development of a structural database dedicated to protein-protein interactions: 2P2IDB (http://2p2idb.cnrs-mrs.fr/). The interface descriptors of protein-protein interfaces which are typical of complexes present in 2P2IDB have been used to develop a qualitative scoring function to assess the ‘druggability' of PPI targets.In the second part of my PhD, I contributed to the analysis of the chemical space of PPI inhibitors present in the 2P2I database using chemoinformatics tools. We defined the ‘Rule-of-4' as a guideline to characterize these compounds. We have used support vector machine approaches to elaborate a protocol: 2P2IHUNTER, which allows filtering large collection of compounds to design chemical libraries dedicated to PPI targets.

Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

<p dir="ltr">Ebola virus is an enveloped filamentous shaped RNA virus which causes severe hemorrhagic fever in humans. Multiple outbreaks of different strains of ebolavirus have been reported in the past years with limited therapeutics available for treatment. Despite some advances in treatment, there remains a lack of knowledge about the mechanisms of ebolavirus replication in host cells.</p><p dir="ltr">Ebolavirus encodes for seven structural proteins with matrix protein (VP40) being the most abundantly expressed viral protein. VP40 is essential for viral assembly and budding as expression of VP40 alone is sufficient for formation of virus-like particles (VLPs). VP40 also disassembles during viral entry to help in viral and host cell membrane fusion. Oligomerization of VP40 has been reported to decrease viral replication and transcription. VP40 can perform these diverse functions by virtue of changes in conformation and oligomerization state. VP40 predominantly exists as a dimer through hydrophobic interactions between the alpha helices of the two protomers. Furthermore, VP40 oligomerizes into a hexamer which serves as the structural unit for cylindrical matrix layer formation. VP40 also forms a ring-shaped octamer for regulation of viral transcription. The different oligomeric forms of VP40 exist in an equilibrium for successful viral infection. However, the exact mechanism of formation, stability, and energetics of conversion between these oligomeric forms is unknown.</p><p dir="ltr">In this study, we performed biophysical analysis on the dimerization interface and identified keystone interactions which when abrogated lead to complete disruption of dimer interface. In addition, peptidomimetics approach was used to design and synthesize a library of compounds to probe the dimerization interface. The compounds were screened using thermal shift assay and then compared using MST and ITC studies. We identified that a peptide mimicking the alpha helical region stabilized by a p-xylene di-cysteine staple was able to bind to VP40 dimer. We also determined that this peptide binds near the dimer interface and was able to slightly shift equilibrium of VP40 dimer towards monomer formation.</p><p dir="ltr">Overall, this report sheds light on critical interactions required for VP40 dimer formation and stability and introduces use of peptidomimetics to probe for VP40 dimerization interface to understand energetics of oligomerization equilibrium, thereby increasing our knowledge about disease mechanism and paving way for development of therapeutics.</p>

The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.

The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein.