Dissertations / Theses on the topic 'Viruses; X-ray crystallography'

The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.

Les virus sont les entités biologiques les plus abondantes dans les océans (∼1031 particules). Ils colonisent tous les écosystèmes de la planète y compris les environnements extrêmement acides, chauds et salins, environnements où les archées sont les organismes dominants. Les virus infectant les Crenarchées hyperthermophiles présentent des morphologies exceptionnelles et aussi une très faible proportion de gènes possédant des homologues avec de fonction connue. Parmi ces virus, le virus ATV (Acidianus two-Tailed virus), infecte les archées hyperthermophiles du genre Acidianus. ATV a la propriété unique de présenter un important développement structural complètement indépendante de son hôte, à l'extérieur de celui-Ci. Les virions d'ATV développent de longues queues à chaque extrêmité de sa capside, mais seulement à des températures proches de celles de l'habitat de son hôte, 85°C. Le sujet de ma thèse a porté sur l'étude structurale de protéines du virion d'ATV. J'ai résolu la structure cristalline de la protéine ATV-273, qui possède un nouveau fold α/β. J'ai aussi déterminé la forme de l'enveloppe de cette protéine par SAXS. J'ai montré qu'il est possible de placer deux dimères d'ATV-273, observés dans la structure cristalline, dans cette enveloppe. Ce résultat est aussi en accord avec l'état d'oligomérisation en solution déterminé par chromatographie d'exclusion stérique couplée à la diffusion de la lumière. La fonction de cette protéine reste cependant inconnue
Viruses are the most abundant biological entity in the oceans (∼1031 particles) and remarkably, viruses populate every ecosystem on the planet including the extreme acidic, thermal, and saline environments where archaeal organisms dominate. The viruses infecting hyperthermophilic Crenarchaea revealed exceptional morphologies and also a very low proportion of genes with recognizable functions and homologues. Among these viruses we find ATV (Acidianus two-Tailed virus). ATV is a virus infecting hyperthermophilic archaea of the genus Acidianus, which has the unique property of undergoing a major morphological development outside and independently of the host cell. Virions develop long tails at each pointed end of the initial lemon-Shaped particle, at temperatures close to those of the host natural habitat, 85 °C. The subject of my thesis has focused on the virion proteins of ATV. I have solved the crystal structure of ATV-273 that revealed a new α/β fold. I have also obtained a SAXS envelope where it is possible to fit two crystal dimers, in agreement with the oligomerization state in solution as determined by size-Exclusion chromatography coupled to multi angle light scattering. The function of this protein, however, could be not determined. Moreover, a negative staining electron microscopy model was obtained for the AAA+ ATPase ATV-618, which belongs to the MoxR familiy and presents sequence high similarity with the AAA-ATase RavA from Escherichia coli K12. I have shown that this thermostable AAA-ATPase enzyme assumes a hexameric ring organisation in the presence of ATP

Viruses populate virtually every ecosystem on the planet. Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world. However, compared to eukaryotic and bacterial viruses, our knowledge of viruses infecting the archaea is limited. Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, structural studies can reveal distant evolutionary relationships and provide functional insights that are not apparent from the primary amino acid sequence alone. Several such structural studies have already contributed to our understanding of the Sulfolobus Spindle-shaped viruses (Fuselloviridae). Here we report the structure of two proteins, SSV1 F112 and SSVRH D212. Biochemical, proteomic and structural studies of F112 reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Continuing these efforts, a second structure was also determined where the overall fold and conservation of active site residues place D212 within the PD-(D/E)XK nuclease superfamily. Notably, the structure of F112 contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses and the evolutionary implications of such distribution are discussed. Here we review and describe our progress towards understanding these viruses at a molecular level.

Sulfolobus turreted icosahedral virus (STIV) was isolated from acidic hot springs of Yellowstone National Park and was the first hyperthermophilic virus described with icosahedral capsid architecture. Structural analysis of the STIV particle and its major capsid protein suggests that it belongs to a lineage of viruses that predates the division of the three domains of life. Functional predictions of the viral proteins are hindered because they lack similarity to sequences of known function. Protein structure, however, may suggest functional relationships that are not apparent from the sequence. Thus, we have initiated crystallographic studies of STIV and expect to gain functional insight into its proteins while illuminating the viral life cycle. These studies may also provide genetic, biochemical, and evolutionary insight into its thermoacidophilic host and the requirements for life in these harsh environments. The first three proteins studied in structural detail are A197, B116, and F93. As anticipated, these structures suggest possible functions. The structure of A197 reveals a glycosyltransferase GT-A fold. Within the context of the GT-A fold, are the canonical DXD motif and a putative catalytic base, hallmarks of this family of enzymes, strongly suggesting glycosyltransferase activity for A197. B116 is a unique structure that lacks significant homology to known protein structures. However, sequence similarity to proteins from other hyperthermophilic viruses reveals conserved surface features suggesting interaction with a host macromolecule, likely DNA. The F93 structure reveals a winged-helix fold common among DNA-binding proteins, in particular, the MarR-like family of transcriptional regulators. The most likely role for F93 is thus regulation of viral transcription. Interestingly, B116 contains an intramolecular disulfide bond while F93 contains an intermolecular disulfide bond. The presence of these disulfide bonds was not anticipated because these proteins are expected to be localized within the host cell. This prompted analysis of the cysteine distribution in the STIV genome, which suggests that disulfide bonds are common in intracellular (cytoplasmic) proteins encoded by STIV. This work is in accordance with accumulating evidence that disulfide bonds are common stabilizing elements in the intracellular proteins of thermophilic organisms in general, and extends the observation to genomes of hyperthermophilic viruses.

Le virus de Chikungunya (CHIKV) est un alphavirus émergent, transmis par les moustiques, qui a provoqué des épidémies de maladies débilitantes chez homme pendant les dernières cinq années. L'invasion de CHIKV dans les cellules sensibles est médiée par deux glycoprotéines virales, E1 et E2, qui portent respectivement les boucles de fusion à la membrane et les déterminants antigéniques principaux, et forment une couche protéinique icosaédrique à la surface du virion. La glycoprotéine E2, provenant du clivage par la furine du précurseur p62 (en E3 et E2), est responsable de la liaison au récepteur, tandis que E1 est impliqué dans la fusion membranaire. Dans le cadre d'un effort multidisciplinaire pour comprendre la biologie de CHIKV, nous avons déterminé les structures cristallines de l'hétérodimère précurseur immature (p62-E1; 2. 17 A de résolution) et du complexe mature (E3-E2-E1; 2. 6 A de résolution). Les structures atomiques nous ont permis de faire la synthèse d'une multitude de données génétiques, biochimiques, immunologiques et de microscopie électronique accumulées pendant plusieurs années sur les arbovirus en général. Cette analyse donne une image détaillée de l'architecture fonctionnelle de la couche de surface (25 MDa) des alphavirus. Les structures des complexes matures et immatures de CHIKV a aussi permis de décrire les causes et les mécanismes du changement de conformation des protéines d'enveloppe du virion lors du passage de celui-ci dans l'endosome à pH acide et précédant la fusion membranaire
Chikungunya is an emerging mosquito-bome alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor to E3 and E2 is responsible for receptor binding and is the major viral antigen. The E1 protein is responsible for inducing the fusion of viral and cellular membranes in the target cell endosome which is required for release of the viral nucleocapsid into the cytoplasm to initiale infection of a cell. While the structure of E1 has been determined, the structure of E2"has remained elusive over the years. This thesis reports the atomic structures of the mature (E3/E2/E1) and immature (P62/E1) envelope glycoprotein complexes from Chikungunya virus determined by X-ray crystallography using a recombinant protein construct. This construct contained the covalently linked ectodomains of p62 and E1. Diffracting crystals of the purified complexes were obtained at neutral pH when the linker joining the ectodomains was cleaved. The glycoprotein structures were fit into reconstructions of the alphavirus virion obtained from cryo-electron microscopy (cryoEM). This analysis resulted in an inferred atomic model of the entire 25MDa surface of the highly conserved alphavirus virion and allowed for the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general

Viruses are a diverse genera of organisms adapted to thrive in many different hosts from prokaryotic to eukaryotic. We present here the structure of bacteriophage PRR1 virus-like particle (VLP), belonging to Leviviridae family. Our structure reveals calcium ions in the VLP. Metal ions are rare in the VLP among the Leviviridae and the calcium ions were found to affect VLP stability. Gene expression in Leviviridae is controlled by a specific interaction between the viral coat protein that assembles to create the VLP, and the genomic RNA. This interaction has been thoroughly studied for the levivirus MS2 but other structural data are scarce. We have solved the structure of PRR1 VLP in complex with its RNA operator stem-loop. Binding of the stem-loop in PRR1 shows similarities to MS2 but also a different arrangement of the nucleotides, in the area of the loop that we could interpret, compared to MS2. The structures of PRR1 increase our knowledge about translational control in Leviviridae and add new information about particle stability within this family. The other virus we investigated is the more infamous human pathogen, the HIV. Because of the high mutation rate of HIV new drugs are needed on a continuous basis. We describe here the structure of two new protease inhibitors bound to the HIV-1 protease and compare them with two previously published inhibitors. Due to an extended P1´site the new compounds are able to exploit a new interaction to Phe53 in the protease structure.

Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 724

In humans, noroviruses (NVs) cause acute epidemic and viral gastroenteritis. NVs do not only infect humans; viruseshave also been found in pigs, cows, sheep, mice and dogs. The focus in this project has been on the murine norovirus(MNV). MNV is a member of the viral family Caliciviridae and it consists of a single-stranded, positive sense RNAgenome. The genome includes three open reading frames (ORFs), ORF1 encodes for a polyprotein that consists of theprecursor to the 6-7 non-structural (NS) proteins. The polyprotein is cleaved by the NS6 protease. The NS6 isresponsible for all the cleaving in ORF1 and that makes it an attractive target for antiviral drugs. The NS6 proteinstructure has been determined at 1.66 Å resolution using X-ray diffraction techniques. Surprisingly, the electrondensity map revealed density for a peptide bound in the active site. The peptide had a length of 7 residues andoriginated from the C-terminus of another chain in an adjacent asymmetric unit. The active site triad was composed ofthe conserved residues; histidine 30, aspargine 54 and cysteine 139, however in the structure the cysteine 139 ismutated to an alanine to inactivate the protease. Activity assays were performed to probe the importance of the residuein position 109 in the β-ribbon located close to the active site. The three full-length constructs with the mutations;I109A, I109S and I109T were found to have less activity than the full-length wt (1-183). A truncated protease, lacking9 residues in the C-terminus, also had less activity. This indicates that the terminal residues are also important foractivity.

Les rétrovirus sont un enjeu de santé publique, aussi bien humaine qu'animale. La compréhension des déterminants structuraux sous-jacents à la fonction de leurs protéines constitue une étape essentielle dans le développement de stratégies antirétrovirales efficaces.Ce manuscrit porte sur l'étude des bases structurales des mécanismes moléculaires impliqués dans les fonctions clés des rétrovirus que sont i) la régulation de l'expression des protéines de rétrovirus complexes et ii) l'assemblage des particules virales, à travers l'étude de deux protéines rétrovirales d'intérêt thérapeutique : la protéine Tax du virus T-lymphotrope humain (HTLV) et la protéine de capside du virus de l'immunodéficience féline (FIV). L'étude structurale de ces deux protéines d'intérêt et la compréhension des mécanismes moléculaires nécessaires à leurs fonctions permettraient d'ouvrir la voie à la conception de nouvelles stratégies antirétrovirales.Malgré de nombreux tests d'expression et de purification, l'étude structurale de la protéine Tax du HTLV n'a pu être réalisée, en raison de son insolubité. Cependant, ce travail doctoral a permis de résoudre, pour la première fois, la structure cristallographique de la protéine de capside entière du FIV. Bien que cette dernière adopte un repliement similaire aux autres capsides rétrovirales dont la structure est connue, elle présente également des spécificités structurales dont les conséquences fonctionnelles seront discutées
Retroviruses are a major concern of public health in humans but also in animals. A better understanding of the structural determinants underlying the functions of retroviral proteins is a crucial step for the development of efficient antiretroviral therapies.This manuscript studies the structural basis of the molecular mechanisms implicated in key functions of retroviruses such as, i) the regulation of complex retroviruses protein expression and ii) the assembly of viral particles, through the study of two retroviral proteins of therapeutic interest: the human T-lymphotropic virus (HTLV) Tax protein and the feline immunodeficiency virus (FIV) capsid protein. The functional and structural studies of these two proteins and the understanding of the molecular mechanisms required for their functions will pave the way to the conception of new antiretroviral therapeutic strategies.Despite several expression and purification assays, no structural studies could be performed for the HLTV Tax protein. However, this study allowed the resolution of the first structure for the full-length FIV capsid protein by X-ray crystallography. Although the FIV capsid protein displays a standard a-helical topology like other retroviral CAs, it also harbors original features whose functional consequences will be discussed

Hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma worldwide. HCV-neutralizing antibody AP33 recognizes a linear, highly conserved epitope on the viral entry protein E2, disrupting the interaction with the cellular receptor CD81 that leads to viral entry. AP33-related anti-idiotypes (Ab₂s) have the potential to carry the internal image of the antigen E2, eliciting the production of AP33-like antibodies in humans. This study reports the mid-resolution structure of the Fab fragment of anti-idiotype A164.3 and the high-resolution structure of the Fab fragment of AP33 in complex with the Fv fragment of anti-idiotype B2.1A. Analysis of the structures and comparison with the previously published structure of AP33 in complex with a peptide corresponding to the E2 epitope, suggests that while A164.3 does not mimic the antigen E2, B2.1A is characterized by high surface complementarity with AP33 and functional antigen mimicry. Thus, B2.1A can be classified as an Ab₂-β, a subgroup of anti-idiotypes carrying the internal image of the antigen. Preliminary binding studies show that AP33 binds B2.1A with nanomolar affinity, supporting the role of B2.1A as an idiotypic vaccine candidate. Zaire ebola virus causes severe, often lethal hemorrhagic fever in humans. Ebola virus polymerase cofactor VP35 is a multifunctional protein involved in, among other functions, dsRNA binding and inhibition of the host's interferon pathways. VP35 contains an N-terminal oligomerization domain and a C-terminal dsRNA-binding domain (RBD). Preliminary results on the oligomerization domain of VP35 suggest that this region contains a coiled-coil motif, as previously reported. In order to validate a recently-discovered dsRNA end-capping pocket as a drug target, the structure of VP35 RBD I278A mutant was solved at high resolution, showing that even a small perturbation in the binding pocket can cause dramatic binding impairment due to loss of contacts with dsRNA.

Depuis sa découverte il y a plus de 30 ans, le Virus de l’Immunodéficience Humaine est à l’origine d’une importante mortalité dans le monde. De par la difficulté de tester l’efficacité de formulations thérapeutiques et/ou vaccinales directement chez l’homme, des études d’infections modèles du VIH, comme celle du Virus de l’Immunodéficience Féline (FIV), ont été entreprises ces dernières années. Au-delà de son intérêt vétérinaire, l’étude du FIV représente un avantage important pour trouver un moyen de contrôler les infections par les lentivirus tel que le VIH. Elle peut permettre de développer et surtout de tester l’efficacité des vaccins et/ou thérapies spécifiques chez le chat, dont le SIDA mime les symptômes et les modifications hématologiques rencontrés chez l’homme. Ce manuscrit s’est intéressé à l’étude structurale de deux familles de protéines virales de ces virus, les protéines lentivirales précoces (protéine Tat du VIH) et tardives (domaines Capside CA et Matrice MA de Gag du FIV). L’étude structurale de ces protéines et leur compréhension fonctionnelle au sein de l’hôte pourront à l’avenir ouvrir de nouvelles voies thérapeutiques et/ou vaccinales contre les lentivirus, palliant ainsi les problèmes existants de résistances virales
Since its discovery 30 years ago, the Human Immunodeficiency Virus is the cause of an important mortality worldwide. Because of the difficulty to test the efficiency of therapeutical and/or vaccinal formulations directly in humans, studies of models of HIV infections, such as the Feline Immunodeficiency Virus (FIV), have been performed in recent years. In addition to its veterinary interest, the study of FIV is an important issue to find a way to control infections by lentiviruses such as HIV. It can help to develop and test the efficiency of specific therapies and/or vaccines for cats, where AIDS mimics the symptoms and hematologic changes observed in humans. This manuscript describes the structural study of two types of viral proteins of these viruses, early lentiviral proteins (HIV Tat protein) and late lentiviral proteins (CA capsid and MA Matrix domains of FIV Gag). The structural study of these proteins and their functional understanding into the host will open new therapeutic and/or vaccine strategies against these lentiviruses in the future, in order to overcome the existing problems of viral resistance

La proteine centrale de la polyproteine gag du virus vih-1, la proteine p24, constitue la capside du virus, une enveloppe conique qui protege l'arn viral. Les structures des domaines n- et c-terminaux constituant la proteine p24 ont ete revelees separement par des travaux de rmn et de cristallographie des rayons x. Cette these presente la determination structurale, par cristallographie des rayons x, de la proteine p24 entiere en complexe avec un fragment d'anticorps. Le fab 13b5 est un fragment d'anticorps monoclonal de souris de type igg1k, dirige contre le domaine c-terminal de la proteine recombinante de p24. L'adnc codant pour le fab 13b5 a ete clone et sequence. Le fab 13b5 a ete cristallise, sa structure a ete determinee par la technique du remplacement moleculaire, puis affinee a 1,8a, jusqu'a un facteur rlibre de 26,8% et un facteur r de 23,7%. Par ailleurs, la structure du complexe fab 13b5-p24 a ete resolue par remplacement moleculaire avec les fragments connus de la proteine p24 ainsi que le fab 13b5 libre a partir de donnees cristallographiques jusqu'a 4a. L'etude de la structure du complexe fab 13b5-p24, affinee jusqu'a 3a de resolution, a permis : de determiner la structure entiere de la proteine p24, de montrer sa flexibilite interne qui est localisee a la region liant les domaines n- et c-terminaux, et d'observer un nouveau type de dimerisation p24-p24, le domaine n-terminal d'une molecule interagissant avec le domaine c-terminal de sa voisine. Cette structure a apporte aussi des informations sur la reconnaissance de la proteine p24 par ce fragment d'anticorps et en particulier a montre que la surface du fab reconnaissant l'antigene est plutot petite avec seulement 4 cdr impliques (h1, h2, h3 et l1) et formee a 82% par des acides amines de la chaine lourde. Finalement, la structure du fab 13b5 seul a 1,8a de resolution, a permis, par comparaison avec celle du fab 13b5 lie a son antigene (dans la structure du complexe), d'observer les changements structuraux du fab 13b5 sous l'effet de la complexation avec son antigene : les cdr-h1 et -h2 montrent quelques adaptations localisees aux chaines laterales des acides amines ; le cdr-h3 est plus largement deplace. On observe, au niveau de la structure quaternaire du fab 13b5, une rotation de 8 de la chaine legere et de la chaine lourde, l'une par rapport a l'autre dans le dimere variable (fv).

Viral proteases play crucial roles in the life cycle and maturation of many viruses by processing the viral polyprotein after translation and in some cases cleaving host proteins associated with the immune response. The essential role of viral proteases makes them attractive therapeutic targets. In this thesis, I provide an introductory summary of viral proteases, their structure, mechanism, and inhibition, while the breadth of this thesis focuses on the Hepatitis C virus (HCV) NS3/4A and Zika virus (ZIKV) NS2B/NS3 viral proteases. HCV NS3/4A protease inhibitors (PIs) have become a mainstay in combination therapies. However, drug resistance remains a major problem against these PIs. In this thesis, I applied insights from the HCV substrate envelope (SE) model to develop strategies for designing PIs that are less susceptible to resistance. Also, I used the HCV NS3/4A protease as a model system to decipher the molecular mechanism and role of fluorination in HCV PIs potency and drug resistance. The drug design strategies described in this thesis have broad applications in drug design. The ZIKV is an emerging global threat, and currently, with no treatment available. In this thesis, I described the discovery, biochemical and antiviral evaluation of novel noncompetitive quinoxaline-based inhibitors of the ZIKV NS2B/NS3 protease. The inhibitors are proposed to interfere with NS2 binding to NS3, thereby preventing the protease from adopting the closed and active conformation. The inhibitors from this work will serve as lead compounds for further inhibitor development toward the goal of developing antivirals.

Ribonuclease HI (RNase HI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. The enzyme has roles in replication, genome maintenance, and is the C-terminal domain of retroviral multi-domain reverse transcriptase (RT) proteins. Murine Leukemia Virus (MLV) and Human Immunodeficiency Virus (HIV) are two such retroviruses and their RNase HI (RNHI) domains are necessary for viral replication, making it an attractive drug target. RNase HI has a “handle region”, an extended loop with a large cluster of positive residues, that is critical for substrate recognition. MLV-RNHI is active in isolation and contains a handle region, but, HIV-RNHI is inactive in isolation and does not contain a handle region. HIV-RT, however, has a region in its polymerase domain (positive charge cluster and aromatic cluster) that makes contact with the RNHI domain that may be serving as a “pseudo” handle region; additionally, insertion of a handle region into isolated HIVRNHI restores its activity. Overall, a breadth of information exists on this region’s dynamics, but important gaps remain unfilled; gaps that may potentially lead to creating effective drugs to treat the above-mentioned viruses. Solution-state nuclear magnetic resonance (NMR) spectroscopy combined with Molecular Dynamic (MD) simulations suggest a model in which the extended handle region domain of the mesophilic Escherichia coli RNHI (EcRNHI) populates "open" (substrate-bindingcompetent) and "closed" (substrate-binding incompetent) states, while the thermophilic Thermus thermophilus RNHI (TtRNHI) mainly populates the closed state at 300 K. In addition, an in silico designed mutant Val98Ala (V98A) EcRNHI was predicted to populate primarily the closed state. Understanding the structural features and internal motions that lead RNase HI to adopt these various conformers is of central importance to better understanding RNase HI’s role in retroviral infection. To formulate a comprehensive model on handle region dynamics, an integrative approach of NMR spectroscopy, X-ray crystallography, and MD simulations is employed. The sensitivity to internal conformational dynamics at multiple time scales of NMR spectroscopy, molecular range and resolution of X-ray crystallography, and structural interpretations of dynamic processes by MD simulations create a synergistic trio capable of tackling this issue. First, the in silico 2-state Kinetic model is validated through NMR observables that correlate with the respective conformers, thus serving as experimental analogs. The NMR parameters also correlate with the Michaelis constants (KM) for RNHI homologs and help to confirm the in silico predictions of V98A EcRNHI. This study shows the important role of the handle region in modulation of substrate recognition. It also illustrates the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function. Next, a deeper dive is taken into handle region dynamics, specifically focusing on residue 88 and the impact its identity has on this region. Its sidechain interactions are shown to directly correlate with handle region conformations and helps to amend the originally proposed in silico 2-state Kinetic model. Lastly, looking at RNHI handle region dynamics through an evolutionary lens opens the door to uncovering novel mutations that have been previously overlooked or not identified. Through a phylogenetic analysis, researchers have reconstructed seven ancestral RNHI mutants and three of them have been expressed here. The sequence identity of these three ancestral mutants range from 60-87% to extant homologs and this is reflected by similar peak positions in their 15N HSQC spectra. Requisite experiments to assign the NMR backbone have been completed.

The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs. The present thesis mainly deals with application of the crystallographic techniques for understanding the structure and function of adenylosuccinate lyase (ASL) from bacterial pathogens Salmonella typhimurium and Mycobacterium tuberculosis as well as its non-pathogenic counterpart Mycobacterium smegmatis. Studies were also carried out to understand the structure-function relationship of the protease in the plant virus Sesbania Mosaic Virus (SeMV). The thesis has been divided into six chapters. The first chapter contains an introduction to nucleotide synthesis and ASL superfamily of enzymes known as the aspartase/fumarase superfamily based on the published literature. Chapter 2 provides the details of the techniques used for the investigations presented in this thesis. Chapters 3-5 deal with the structural and functional studies carried out on ASL from the three bacterial organisms. Chapter 6 deals with the simulation studies carried out on SeMV protease. Mechanism and importance of nucleotide synthesis is introduced in Chapter 1, with special emphasis on purine de novo and salvage pathways. ASL is introduced as an important enzyme for purine synthesis. Its superfamily, the aspartase/fumarase superfamily of enzymes is described in detail with respect to its structure, function and pathophysiology. Objectives of the present study are outlined towards the end of the chapter. The experimental and computational techniques utilized during the course of my research are described in Chapter 2. These techniques include gene cloning, protein expression and purification, kinetic and biophysical characterization of proteins, crystallization, X-ray diffraction, data collection and processing, structure solution, refinement, model building, validation and structural analysis, phylogenetic studies, molecular docking and molecular dynamic simulation studies. Adenylosuccinate lyase is an important enzyme participating in purine biosynthesis. With the emergence of drug resistant variants of various pathogens, ASL has been recognized as a drug target against microbial infections. Chapter 3 deals with the structural and functional characterization of ASL from Salmonella typhimurium. Two constructs of the StASL gene were cloned and expressed leading to the purification of truncated (residues 1-366) and full-length (residues 1-456) polypeptides. Crystallization of the two polypeptides resulted in three independent structures. The full-length structure was very similar to the E. coli ASL structure consistent with 95% amino acid sequence identity between the two polypeptides. However, the truncated structures showed large distortions, especially of the active site residues, accounting for the catalytic inactivity of the truncated polypeptide in spite of retaining all residues considered important for function. The full-length ASL was catalytically active. A unique feature observed in StASL, not reported in other ASLs, was its allosteric regulation by the substrate. Kinetic studies also revealed hysteretic behavior of the enzyme. The electron density map of the full-length structure showed two novel densities on the molecular 2-fold axis into each of which a molecule of cadavarine could be fitted. Docking studies revealed a ligand-binding site at the inter-subunit interface between the two observed densities which might represent a potential allosteric site. Combining the structural and kinetic results, a possible morpheein model of allosteric regulation of StASL was hypothesized. Chapter 4 deals with the crystallographic and kinetic investigations on ASL from Mycobacterium smegmatis and Mycobacterium tuberculosis. MsASL and MtbASL were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at 2.16 Å resolution. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. Most of the active site residues were found to be conserved with the exception of Ser 148 and Gly 319 of MsASL. Ser 148 is structurally equivalent to a threonine in most other ASLs. Gly 319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active when compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria, their high GC containing genomes as well as with their dependence on other salvage pathways for the supply of purine nucleotides. Chapter 5 deals with the identification of the catalytic residues important for ASL catalysis in view of the earlier conflicting reports on the identity of these residues. pH-dependent kinetic studies were performed on full-length StASL. The theory behind these studies is also described in this chapter. Two residues with pKa values of 6.6 and 7.7 were identified as essential for the enzymatic activity. These results were interpreted along with structural comparison of MsASL and other superfamily enzymes with ordered C3 loops. They suggest that His 149 and either Lys 285 or Ser 279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. The final Chapter 6 of the thesis deals with the structural and dynamic studies carried out on Sesbania mosaic virus (SeMV) protease. The chapter begins with a general introduction to viruses, followed by a brief summary of SeMV. The goal of this study is to understand the interactions between the protease and VPg at a structural level using the information available from biochemical studies. Crystallographic studies initiated for the mutant H275APro and Y315APro were unsuccessful due to the insolubility of the proteins. Co-crystallization or soaking experiments of wild type protease with cognate peptides were unsuccessful due to the inability of the enzyme to bind to its substrates in the absence of VPg. Higher resolution structure of wild type protease did not yield any new insights when compared to the earlier reported structure determined at a lower resolution. In the absence of structural insights, molecular dynamic simulations were carried out on wild type protease structure and in silico generated mutants using GROMACS package. The studies showed the importance of flipping of residue Phe 301 and opening-closing of the loop region corresponding to residues 301-308 for the catalytic mechanism. The thesis concludes with Future perspectives of the various studies carried out on ASL and SeMV protease. The atomic coordinates determined from the work presented in this thesis have been deposited in the PDB and the assigned PDB codes are reported in the respective chapters. Publications cited in the thesis are listed in the Bibliography section.

A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping. This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations. Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design. Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed. SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided. Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed. Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants. Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains. Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations. Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis. In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.