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Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

Pantothenate kinase (PanK) is an ubiquitous and essential enzyme that catalyzes the first step in the universal Coenzyme (CoA) biosynthesis pathway. In this step, pantothenate (Vitamin B5) is converted to 4′-phosphopantothenate, which subsequently forms CoA in four enzymatic steps. In bacteria, three types of PanK’s have been identified which exhibit wide variations in their distribution, mechanisms of regulation and affinity for substrates. Type I PanK is a key regulatory enzyme in the CoA biosynthesis pathway and its activity is feedback regulated by CoA and its thioesters. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on type I PanK from Mycobacterium tuberculosis (MtPanK) was initiated and the structure of this enzyme in complex with a CoA derivative has been reported earlier. To further elucidate the structural basis of the enzyme action of MtPanK, several crystal structures of the enzyme in complex with different ligands have been determined in the present study. In conjunction to this, solution studies on the enzyme were also carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion method was used for crystallization in all cases. The X-ray intensity data were collected using a MAR Research imaging plate system mounted on a Rigaku RU200 and Bruker-AXS Microstar Ultra II rotating anode X-ray generator. The data were processed using the HKL and MOSFLM and SCALA from the CCP4 suite. The structures were solved by the molecular replacement method using the program AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT and the refined structures were validated using PROCHECK and MOLPROBITY. Secondary structure was assigned using DSSP, structural superpositions were made using ALIGN and buried surface area was calculated using NACCESS. Solution studies on CoA binding and catalytic activity were carried out using Isothermal titration calorimetry (ITC). To start with, the crystal structures of the complexes of MtPanK were determined with (a) citrate, (b) the non-hydrolysable ATP analog AMPPCP and pantothenate (initiation complex), (c) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (end complex), (d) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (intermediate complex), (e) CoA, (f) ADP prepared by soaking and co-crystallization, which turned out to have identical structures and (g) ADP and pantothenate. Unlike in the case of the homologous E.coli enzyme (EcPanK), AMPPCP and ADP occupied different, though overlapping, locations in the respective complexes; the same was true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK was found to be substantially preformed while that of EcPanK exhibited considerable plasticity. The difference in the behavior of the E.coli and M.tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action and the changes in the locations of ligands exhibited by M.tuberculosis pantothenate kinase are remarkable and novel. The movement of ligands exhibited by MtPanK during enzyme action appeared to indicate that the binding site of the enzyme was less specific for a particular type of ligand than EcPanK. Kinetic measurements of enzyme activity showed that MtPanK had dual substrate specificity for ATP and GTP, unlike the enzyme from E.coli which showed a much higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes was provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate (2) GDP and phosphopantothenate (3) GDP (4) GDP and pantothenate (5) AMPPCP and (6) GMPPCP and the structures of the complexes of the two enzymes involving CoA and different adenyl nucleotides. The explanation was substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. Dual specificity of the type exhibited by this enzyme is rare and so are the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands and specificity. The crystal structures of MtPanK in binary complexes with nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) provided insights about the natural location and conformation of nucleotides. In the absence of pantothenate, the NDP and the NTP bound with an extended conformation at the same site. In the presence of pantothenate, as seen in the initiation complexes, the NTP had a closed conformation and an altered location. However, the effect of the nucleotide on the conformation and the location of pantothenate were yet to be elucidated as the natural location of the ligand in MtPanK was not known. This lacuna was sought to be filled through X-ray analysis of the binary complexes of MtPanK with pantothenate and two of its derivatives, namely, pantothenol and N-nonyl pantothenamide (N9-Pan). These structures demonstrated that pantothenate, with a somewhat open conformation occupied a location similar to that occupied by phosphopantothenate in the “end” complexes, which was distinctly different from the location of pantothenate in “closed” conformation in the ternary “initiation” complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appeared to play a critical role in the movement of ligand that took place during enzyme action. The structure analysis of the binary complexes with the vitamin and its derivatives completed the description of the locations and conformations of nucleoside di and triphosphates and pantothenate in different binary and ternary complexes. These complexes provide snapshots of the course of action of MtPanK.

Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis. The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures. Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth. The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule. After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis. In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.