Thymidylate kinase (TMK) is a key enzyme for DNA synthesis. It occurs at the junction of the de novo and salvage pathways for the synthesis of deoxythymidine triphosphate (dTTP). Its inhibition affects cell viability, thereby making it an important target for the development of anticancer, antibacterial and antiparasitic drugs. This thesis describes the analyses of the sequence, structure and dynamics of thymidylate kinase to obtain insights into its function. Two thermophilic variants of the enzyme were chosen for our studies. The studies provide valuable insights about the active site residues and the mechanism of catalysis, which have implications in protein engineering and design of specific inhibitors. Following is a chapter-wise description of the overall layout of the thesis. Chapter 1 | Introduction: This chapter provides a brief survey of the literature on TMKs and the scope of the work presented in the thesis. TMK belongs to the nucleoside monophosphate kinase (NMPK) family of enzymes, which includes adenylate kinase (AMK), guanylate kinase (GMK), uridylate kinase (UMK) and cytidylate kinase (CMK). The NMPK family of enzymes is associated with the reversible transfer of the terminal phosphoryl group from a nucleoside triphosphate (NTP) (usually adenosine triphosphate, i.e., ATP) to a nucleoside monophosphate (NMP). The identity of the NMP substrate varies among different enzymes. NMPKs share a common Rossmann fold and are comprised of a conserved P-loop, Lid region, CORE and NMP domains. The enzymes in the NMPK family also contain structurally similar active site architecture. Besides the three signature motifs, there are other conserved residues at the active site of TMK which are involved in interactions with the substrates ATP and dTMP. Despite the overall similarity, TMKs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the residues responsible for these differences have not been studied. This thesis describes a comprehensive analysis of the sequence space of TMKs to detect the residues involved in such diversity. Subsequently, TMKs from a thermophilic archaeon (Sulfolobus tokodaii) and a hyperthermophilic bacterium (Aquifex aeolicus) were chosen for biochemical characterization and structural studies. Of these, the Sulfolobus tokodaii TMK (StTMK) has low sequence identity to the other TMKs of known three dimensional (3D) structures. Crystal structure analyses depicted the presence of some novel structural features and provided insights into the role of a conserved Arginine residue in function, which was verified through computational studies and mutagenesis experiments. Finally, the study on Aquifex aeolicus TMK resulted in multiple crystal structures of the apo form and different holo forms. These helped us to understand the mechanistic details of TMK-mediated catalysis, namely, the order of substrate binding and the reaction mechanism for phosphate transfer. Chapter 2 | Materials and Methods: This chapter provides a brief description of the procedures used to carry out the thesis work. The protein samples were purified to a high degree using column chromatography, and the purity was assessed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. Circular dichroism (CD) spectroscopy was employed to assess if the purified protein was well-folded. The pure and properly folded protein samples were used in further experiments. Differential scanning fluorimetry (DSF) was performed to determine the melting temperature of the thermophilic protein. MicroScale Thermophoresis (MST) and Surface Plasmon Resonance (SPR) were carried out to detect protein-substrate interactions. The protein samples were crystallized using the hanging drop vapour diffusion and microbatch under-oil techniques using commercially available crystallization screens, and the conditions which gave crystals were further optimized. Diffraction data, collected at either the home source or the synchrotron, were processed and scaled. Subsequently, phase information was obtained using the molecular replacement (MR) calculations. The MR solution was refined till convergence and its geometry was validated using different softwares. Finally, molecular dynamics (MD) simulations were performed to study the functionally important motions in the protein. Chapter 3 Insights into substrate specificity and oligomerization mode of Thymidylate Kinases from sequence evolution and conformational dynamics: Thymidylate kinase homologs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on thymidylate kinase from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our sequence analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs, which was also indicated in MD simulations on the protein 3D structure. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode and substrate recognition in thymidylate kinases and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors. Chapter 4 | Biochemical and Structural characterization of a thermophilic variant of thymidylate kinase: This chapter reports the biochemical characterization and crystal structure determination of thymidylate kinase from the hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. The different structures had resolution ranging between 1.60 Å and 2.40 Å. StTMK is a thermostable enzyme with a melting temperature of 85.3 °C, as observed from thermal unfolding studies. The protein exists as a dimer in solution. A coupled enzyme assay, performed using thermo-stable lactate dehydrogenase (TtLdh) and pyruvate kinase (TtPk) from Thermus thermophilus, showed that StTMK has optimum activity at 80 °C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. A novel insertion (9 residues long) is observed in the C-terminal stretch of the Lid region. However, it is relatively rigid, which may be attributed to the presence of two proline residues and a hydrogen bond with an arginine residue in the α4/α5 loop. The C-terminus of the α2 helix points away from the Lid region in StTMK to avoid steric clashes with the Lid insertion. The main chain dihedral angles of the conserved Arg in the DRX motif are in the disallowed region of the Ramachandran plot in all holo TMK structures, wherein it forms several conserved hydrogen bonds with residues in the P-loop, α4 helix and α7 helix, as well as with the phosphate groups of both the substrates. A similar feature is observed in some of the StTMK structures. However, torsion angles in the allowed region of the Ramachandran plot are observed in one chain each in two of the apo structures. Further, conformational rearrangements of this Arg and its neighboring residues at the binding site of the second substrate are observed. The functional implication of this variation is described in the next chapter (chapter 5). Chapter 5 | Role of a conserved active site Arginine residue in Thymidylate kinase: Analysis of the structures of StTMK revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Conformational heterogeneity of Arg can also be observed in some structures of Staphylococcus aureus and human TMK. However, the functional implication of this feature has not been probed before. The rearrangements of Arg93 are accompanied by related changes in the conformations of its neighbouring residues at the active site. This leads to three distinct conformational states in the dTMP-binding site, namely ‘Arg in’, ‘Arg intermediate’ and ‘Arg out’. Only the ‘Arg in’ state was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. This is hindered in the ‘Arg out’ and ‘Arg intermediate’ states. MD simulations showed that the torsion angles of the DRX Arg can sample between allowed and disallowed values in the apo-protein, with a preference for the catalytically suitable disallowed conformation in the holo-protein. Computational alanine scanning and MM/PBSA binding energy calculation further revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Our work provides the first experimental evidence for the functional importance of Arg93 and gives insight into its regulatory role in the catalytically competent placement of dTMP. Our study also has implications for the development of potent inhibitors to lock the enzyme in the catalytically non-productive state. Chapter 6 | Characterizing active site dynamics from structural studies on the Intermediates along the reaction coordinate of a hyperthermophilic Thymidylate Kinase: TMK belongs to the family of nucleoside monophosphate kinases (NMPKs), several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild type protein and mutants. Further, the enzyme was found to exhibit broad specificity towards phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes. Finally, the implications of the work described in this thesis and its future applications have been discussed in the section titled ‘Future prospects’. The work described in chapters 3 – 6 have been published in peer reviewed journals. Additionally, the author was involved in several collaborative projects which also resulted in publications (reprints attached in appendix).

Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method. An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS. 23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.

Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.

The N-methyl-D-aspartate receptor (NMDAR) hypofunctional hypothesis is believed to explain one of the contributing factors to schizophrenia. This hypothesis suggests the dysregulation of NMDAR, a protein responsible for receiving signals from the synapses between neurons, is the cause of some of the symptoms seen in schizophrenia. The post synaptic density protein 95 (PSD95) uses its PDZ-domains to help facilitate the received signal from NMDAR to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) which in turn transmits the signal through the neuron. One way to increase the function of NMDAR could be to increase its affinity towards PDZ-domains of PSD95 using a small molecule. Fragment based drug design (FBDD) is one way to screen for molecules that modulates the NMDAR-PDZ interaction. This work describes the development of differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR) assays using a fusion protein to screen for molecules that potentiate the interaction between NMDAR and AMPAR as well as methods assisting in the prioritization of hits based on both affinity, selectivity, and mechanism. The developed assays were used to screen a library containing 768 compounds. Screen positives and other compounds of interest were triaged and evaluated based on affinity, selectivity, and ability to modulated peptide binding resulting in eight confirmed hits that interacts with the two PDZ-domains of PSD95 investigated. As part of this work, the dissociation constant (KD) was determined for a panel of peptides representing versions of the truncated NMDAR GluN2b-subunit C-terminal towards PDZ1 and 2 of PSD95.