Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below. N1TP + NDK ↔N1DP+ −NDK-His* (1) N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M. smegmatis, being a saprophytic, fast growing and non-pathogenic mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism. The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans. Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from Mycobacterium smegmatis mc 155 The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M. smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK. Chapter 3. Characterisation of Oligomerisation Property of M. smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat and SDS. Chemical cross-linking experiments revealed that MsNDK forms dimer, tetramer and hexamer. Homology modeling of MsNDK on the MtNDK crystal structure supported the existence of hexamer as three homodimers. Gln 17, Ser 24 and Glu 27 were found to be positioned at the dimer interface. Mutations on these residues did not abolish the stability of the respective mutant dimers in the presence of SDS and heat. Modeled structure of MsNDK revealed the existence of hydrophobic interactions at the dimer interface. In silico approach helped in mapping the existence of hydrophobic interactions at the dimer interface as two consecutive β-strands. Exposure of hydrophobic residues, using organic solvent methanol, abolished the dimer completely, indicating the vital role of hydrophobic interactions in the dimer stability. In solution, the native MsNDK was found to be a hexamer. Chapter 4. Mycobacterial Nucleoside Diphosphate Kinase Functions as GTPase Activating Protein for Mycobacterial Cytokinetic Protein FtsZ In Vitro Mammalian, plant, and bacterial NDKs can function as GTPase activating protein (GAP) for small G proteins namely, p21 Ras, Rad, and Rho-GTPases in animals and Pra1, Pra2, and GPA1 in Arabidopsis thaliana in vitro. We examined whether NDK of M. tuberculosis (MtNDK) can function as GAP in vitro for the cytokinetic protein FtsZ of Mycobacterium tuberculosis (MtFtsZ), which is a protein with a classical G-protein fold, possessing GTP-binding and GTPase activities (like G proteins). Both MtNDK and MsNDK could function as GAP for MtFtsZ and FtsZ of M. smegmatis (MsFtsZ) respectively in vitro. Similarly, MtNDK could function as GAP for MsFtsZ and reciprocally MsNDK could function as GAP from MtFtsZ. Interaction of NDK with respective FtsZ could be observed. Physiological implications of GAP activity of NDK on FtsZ are discussed. Chapter 5. Transcriptional Analyses of Nucleoside Diphosphate Kinase Gene of Mycobacterium smegmatis mc 155 Although there are studies on the structural and functional aspects of NDK, there are not many studies available on the transcriptional analysis of nucleoside diphosphate kinase (NDK) gene expression in general and nothing in particular in mycobacterial systems. Therefore we studied the transcriptional analysis of expression of Msndk gene, in order to map the Transcriptional Start Site (TSS), identification of promoter elements, and elucidated of transcriptional activity of the promoters. Expression of Msndk gene was analysed in exponential growth phase and under two different stress conditions wherein DNA replication gets arrested. Hydroxy Urea (HU), which reduce dNTP pools by inhibiting ribonucleotide reductase and Phenethyl Alcohol (PEA), which affects membrane structure resulting in DNA replication arrest, were used. Two transcripts and their promoter elements were mapped and their promoter activities were demonstrated. The profile of transcripts was found to be identical under the three different conditions examined.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.

BIOLOGICAL processes are governed through specific interactions of macromolecules. The three-dimensional structural information of the macromolecules is necessary to understand the basis of molecular recognition. A large number of protein structures have been determined at a high resolution using various experimental techniques such as X-ray crystallography, NMR, electron microscopy and made publicly available through the Protein Data Bank. In the recent years, comprehending function by studying a large number of related proteins is proving to be very fruitful for understanding their biological role and gaining mechanistic insights into molecular recognition. Availability of large-scale structural data has indeed made this task of predicting the protein function from three-dimensional structure, feasible. Structural bioinformatics, a branch of bioinformatics, has evolved into a separate discipline to rationalize and classify the information present in three-dimensional structures and derive meaningful biological insights. This has provided a better understanding of biological processes at a higher resolution in several cases. Most of the structural bioinformatics approaches so far, have focused on fold-level analysis of proteins and their relationship to sequences. It has long been recognized that sequence-fold or fold-function relationships are highly complex. Information on one aspect cannot be readily extrapolated to the other. To a significant extent, this can be overcome by understanding similarities in proteins by comparing their binding site structures. In this thesis, the primary focus is on analyzing the small-molecule ligand binding sites in protein structures, as most of the biological processes ranging from enzyme catalysis to complex signaling cascades are mediated through protein-ligand interactions. Moreover, given that the precise geometry and the chemical properties of the residues at the ligand binding sites dictate the molecular recognition capabilities, focusing on these sites at the structural level, is likely to yield more direct insights on protein function. The study of binding sites at the structural level poses several problems mainly because the residues at the site may be sequentially discontinuous but spatially proximal. Further, the order of the binding site residues in primary sequence, in most of cases has no significance for ligand binding. Compounding these difficulties are additional factors such as, non-uniform contribution to binding from different residues, and size-variations in binding sites even across closely related proteins. As a result, methods available to study ligand-binding sites in proteins, especially on a large-scale are limited, warranting exploration of new approaches. In the present work, new methods and tools have been developed to address some of these challenges in binding site analysis. First, a novel tool for site-based function annotation of protein structures, called PocketAnnotate was developed ( http://proline.biochem.iisc.ernet. in/pocketannotate/). PocketAnnotate, detects the putative binding sites from a given protein structure and compares them to known binding sites in PDB to derive functional annotation in terms of ligand association. Since the tool derives functional annotation at the level of binding sites, it has an advantage over other methods that solely utilize fold or sequence information. This becomes even more important for cases where there is no detectable homology with entries in existing databases, as Pocket Annotate does not depend on evolutionary based information for annotation. Second, a web-accessible tool for in silico almandine scanning mutations of binding site residues called ABS-Scan has been developed ( http://proline.biochem.iisc.ernet.in/abscan/). This tool helps in assessing the contribution of the individual residues of binding sites in the protein towards ligand recognition. All residues, one at a time, in a binding site are mutated systematically to an alanine and the ability of the corresponding mutant to bind a given ligand is analyzed. The contribution of each residue towards ligand binding is calculated through a G value derived by comparing the binding affinity to the wild-type protein-ligand complex. Third, a database called Protein-Ligand Interaction Clusters (PLIC) has been developed to identify and analyze the information of similarity across binding sites in PDB, which has been provided in the form of a web-accessible database ( http://proline.biochem.iisc.ernet/ PLIC). Protein-ligand interactions are primarily explored using three different computational approaches - (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of chemical probes, (ii) atomic contacts between protein and ligands (iii) binding energetics involved in interactions derived from scoring functions developed for docking. The information on variations in these features derived from different computational tools is also included in the database for enabling the characterization of the binding sites. As a case study to demonstrate the usefulness of these tools, they have been applied to decipher the complexity of S-adenosyl methionine interactions with the protein. Around 1,213 binding sites of SAM or SAM-like compounds could be extracted from the PLIC database. The SAM or SAM-like compounds were observed to interact with ∼18 different protein-fold types. The variations in different protein-ligand contacts across fold types were analyzed. The fold-specific interaction properties and contribution of individual residues towards SAM binding are identified. The tools developed and example analyses using them are described in Chapter 2. Chapter 3 describes a large-scale pocketome analysis from structural complexes in PDB, in an effort to characterize the known pocket space of protein-ligand interactions. Tools devel-opted as described in Chapter 2 are used for this. A set of 84,846 binding sites compiled from PDB, have been comprehensively analyzed with an objective of obtaining (a) classification of binding sites, (b) sequence-fold-site relationships among proteins, (c) a minimal set of physicochemical attributes sufficient to explain ligand recognition specificity and (d) site-type specific signatures in terms of physicochemical features. A new method to describe binding sites was developed in the form of BScIds such that the structural fold information is well captured. Binding sites and similarities among them were abstracted in the form of networks where each node represents a binding site and an edge between two nodes represents significant similarity between the sites at the structural level. Pocketome networks were constructed from the large-scale information on protein-ligand interactions in the PLIC database. The large pocketome network was then studied to derive relationships between protein folds and chemical entities they interact with. A classification of the binding pockets was achieved by analyzing the pocketome network using graph theoretical approaches combined with clustering methods. 10,858 clusters were identified from the network, each indicating a site-type. Thus, it can be said that there are about 10,858 site-types. Classification of ligand associations into specific site-types helps greatly in resolving the complex relationships by yielding specific site-type ligand associations. The observed classification was further probed to understand the basis of ligand recognition by representing the pockets through feature vectors. These features capture a wide range of physicochemical properties that can be used to derive site-type specific signatures and explore the pocket-space of protein-ligand interactions. A principal component analysis of these features reveals that binding site feature space is continuous in the entire PDB and minor changes in specific features can give rise to significant differences in ligand specificity, consequently defining their distinct functional roles. The weights were also derived for these features through the use of different information theoretic approaches to explain the multiple-specificity of protein-ligand interactions. Analysis of binding sites arising from contribution of residues from different protein fold-types revealed increasing diversity of physicochemical properties at the site, supporting the hypothesis that combination of folds could give rise to new binding sites. Given that a finer appreciation of the molecular mechanisms within the cell is possible only with the structural information, the next objective was to explore if a structural view of an entire proteome can be obtained and if a pocketome could be constructed and analyzed. With this in mind, the causative agent of tuberculosis - Mycobacterium tuberculosis (Mtb) was chosen. Mtb is also being studied in the laboratory from a systems biology perspective, which enabled exploration of how systems and the structural perspectives could be combined and applied for drug discovery. Chapters 4 to 6 describe this effort. The genome sequence of Mycobacterium tuberculosis (Mtb) H37Rv, indicates the presence of ∼4,000 protein coding genes, of which experimentally determined structures are available for ∼300 proteins. Further, advances in homology modeling methods have made it feasible to obtain structural models for many more proteins in the proteome. Chapter 4 describes the efforts for obtaining the Mtb structural proteome, through which the three-dimensional struc-tures were derived for ∼70% of the proteins in the genome. Functional annotation of each protein was derived based on fold-based functional assignments, binding-site comparisons and consequent ligand associations. PocketAnnotate, a site-based function annotation pipeline was utilized for this purpose and is described in Chapter 2. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides a unique opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 unique folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1,728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. A web-accessible database MtbStructuralproteome has been developed to make the data and the analyses available to the community, ( http://proline.physics.iisc.ernet.in/Tbstructuralannotation). The resource, being one of the first to be based on structure-based functional annotations at a genome scale, is expected to be useful for better understanding of tuberculosis and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well. Chapter 5 describes the characterization of the Mtb pocketome. For the structural models of the Mtb proteome described in chapter 4, a genome-scale binding site prediction exercise was carried out using three different computational methods and subsequently obtaining consensus predictions. The three methods were independent and were based on considering geometry, inter-molecular energies with probes and sequence conservations in evolutionarily related proteins respectively. In all, 13,858 consensus binding pockets were predicted in 2,877 proteins. The pocket space within Mtb was then explored through systematic all-pair comparisons of binding sites. The number of site-types within Mtb was found to be 6,584, as compared to the ∼400 structural folds and 1,831 unique sequence families. This reveals that the pocket space is larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. By comparing the pockets with the PDB sites enclosing known ligands, around 6906 binding sites were observed to exhibit significant similarity in the entire pockets to some or the other known binding site in PDB. 1,213 metabolites could be mapped onto 665 enzymes covering most of the metabolic pathways. The identified ligands serve as a predicted metabolome for unit abundances of the proteins. A list of proteins containing unique pockets is also identified. The binding pockets, similarities they share within Mtb and the ligands mapped onto them are all made available in a web-accessible database at http://proline.biochem.iisc.ernet.in/mtbpocketome/. The availability of structural information of the pocketome at a genome-scale opens up several opportunities in drug discovery. They can be directly applied for understanding mechanism of drug action, predicting adverse effects and pharmacodynamics of a drug. Moreover, it enables exploration of new ideas in drug discovery. Polypharmacology is a new concept that aims at modulating multiple drug targets through a single chemical entity. Currently, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. In this study, a structural-proteomics approach is explored to first characterize the pocketome and then utilize it to identify similar binding sites. The knowledge of similarity relationships between the binding sites within the genome can be used in identifying possible polypharmacological drug targets. A pocket similarity based clustering of binding site residues resulted in identification of binding site sets, each having a theoretical potential to interact with a common ligand. A polypharmacological index was formulated to rank targets by incorporating a measure of drug ability and similarity to other pockets within the proteome. By comparing with known drug binding sites from databases such as the Drug Bank, the study has yielded a ready shortlist that includes sets of promising drug targets with polypharmacological possibilities and at the same time has identified possible drug candidates either directly for repurposing or at the least as significant lead clues that can be used to design new drug molecules against the entire group of proteins in each set. This analysis presents a rational approach to identify targets with polypharmacological potential, clues about lead compounds and a list of candidates for drug repurposing. This thesis demonstrates the feasibility of utilizing the structural bioinformatics approaches at a genome-scale. The tools developed for analyzing large-scale data on protein-ligand inter-actions could be applied to characterize the pocket-space of protein-ligand interactions. The network theory approaches applied in this work, make large-scale data tractable and enable binding-site typing. The binding site analysis at a genome-scale for Mtb is first of its kind and has provided novel insights into the pocket space. The binding site analysis performed on a genome-scale for Mtb provided an opportunity to rationalize the polypharmacological target selection and explore drugs for repurposing in TB. In the larger context, structural modelling of a proteome, mapping the small-molecule binding space in it and understanding the determinants of small-molecule recognition forms a major step in defining a proteome at higher resolution. This in turn will serve as a valuable input towards the emerging field of structural-systems biology, which seeks to understand the biological models at a systems level without compromising on the resolution of the study.

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.

The Mycobacterium genus contains major human pathogens, like Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. They have evolved as successful human pathogens by adapting to the adverse conditions prevailing inside the host, which include host immune activation, nutrient depletion, hypoxia, and so on. During such adaptation for the survival and establishment of persistent infection inside the host, the pathogen, like M. tuberculosis, regulates its cell division. It is known that M. tuberculosis enters a state of non-replicating persistence (NRP) inside the host, to establish latent infection, which helps the survival of the pathogen under adverse host conditions such as hypoxia and nutrient depletion. The pathogen can reactivate itself, to come out of the NRP state, and establish active infection at a later stage, when conditions are suitable for its proliferation. The altered physiological state of the latent bacterium makes it tolerant to drugs, which are only effective against proliferating tubercle bacilli. In view of this unique behavioural physiology of tubercle bacilli, it is important to study the process of cell division and how it is regulated in the NRP and actively growing states. The work reported in the thesis is an attempt to understand these aspects of mycobacterial cell division. iii Chapter 1. Introduction: This chapter gives a detailed introduction to bacterial cell division and its regulation in various organisms, like Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and others. In the background of this information, the major studies on mycobacterial cell division and its regulation are presented. Chapter 2. Materials and Methods: This chapter describes in detail all the materials and methods used in the experiments, which are presented in the four data chapters, 3-6. Chapter 3. Ultrastructural Study of the Formation of Septal Partition and Constriction in Mycobacteria and Delineation of its Unique Features: Mycobacteria have triple-layered complex cell wall, playing an important role in its survival under adverse conditions in the host. It is not known how these layers in the mother cell participate during cell division. Therefore, the ultrastructural changes in the different envelope layers of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium xenopi, during the process of septation and septal constriction, were studied, using Transmission and Scanning Electron Microscopy. The unique aspects of mycobacterial septation and constriction were identified and were compared with those of E. coli and Bacillus subtilis septation. Further, based on all these observations, models were proposed for septation in M. tuberculosis and M. smegmatis. Chapter 4. Identification of Asymmetric Septation and Division in Mycobacteria and Its Role in Generating Cell Size Heterogeneity: Bacterial populations are known to harbour phenotypic heterogeneity that helps survival under stress conditions, as this heterogeneity comprises subpopulations that have differential susceptibility to stress conditions. The iv heterogeneity has been known to lead to the requirement for prolonged drug treatment for the elimination of the tolerant subpopulation. Hence, it is important to study the different mechanisms, which operate to generate population heterogeneity. Therefore, in this chapter, studies were carried out to find out whether asymmetric septation and division occur in mycobacteria to generate cell size heterogeneity. Subpopulations of mycobacterial mid-log phase cells of M. tuberculosis, M. smegmatis, and M. xenopi were found to undergo asymmetric division to generate cell size heterogeneity. The asymmetric division and the ultrastructure and growth features of the products of the division were studied. Chapter 5. Study of Mycobacterial Cell Division Using Growth-Synchronised Cells: In this chapter, different stages of cell septation and constriction were studied using growth-synchronised M. smegmatis cells. Phenethyl alcohol (PEA), which has been found to reversibly arrest mycobacterial cells, was used for growth synchronisation. The growth-synchronised mycobacterial cells, which were released from PEA block, were studied at different stages of septation and septal constriction, at the ultrastructural and molecular levels. Chapter 6. Identification of the Stage of Cell Division Arrest in NRP Mycobacteria: The exact stage at which the NRP tubercle bacilli are arrested in cell division is currently unknown. In Wayne’s in vitro model for hypoxia-responsive tubercle bacilli, gradual depletion of oxygen leads to hypoxic stress, inducing the bacilli to enter non-replicating persistence (NRP) state. Using this model, the stage of cell division arrest in M. tuberculosis was characterised at the ultrastructural and molecular levels. Hypoxia-stressed M. smegmatis was used as an experimental system for contrast. The thesis concludes with salient findings, a bibliography, and the list of publications.