| dc.description.abstract | Genetic material of bacteria is subject to damage due to multitudinous factors, both extrinsic and intrinsic in origin. Mechanisms for the maintenance of genomic integrity are thus essential for a bacterium to survive. Bacterium also requires appropriate minor changes in the genetic material so as to adapt to the changing environments. Structural and related studies of two proteins from mycobacteria, one involved in recombinational DNA repair (RecA) and the other involved in SOS response which helps in adaptation to stress (LexA) form the subject matter of the thesis.
The available literature on structural and related studies on RecA and LexA are reviewed in the introductory chapter. The action of RecA involves transition to an active filament formed in association with DNA and ATP, from an inactive filament in the absence of DNA. The structure of the inactive filament was first established in E. coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP were thoroughly characterised and the DNA binding loops were visualised in this laboratory using the crystal structures involving the proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, was identified. The 20 residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, was defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue was explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. MtRecA has an additional importance as an adjuvant drug target in Mycobacterium tuberculosis. Apart from recombination, another important property of RecA is its coprotease activity whereby it stimulates the inherent cleavage of a certain class of proteins. One of the substrates for the coprotease activity of RecA is LexA. LexA is a transcriptional repressor involved in SOS response in bacteria. LexA performs its function through an autoproteolysis stimulated by RecA, resulting in the derepression of the genes under its control. Structural studies on LexA from E. coli have shown that it has an N-terminal domain involved in binding to DNA and a C-terminal domain involved in catalysis and dimerisation. LexA mediated SOS response in bacteria has been shown in many cases to be responsible for the resistance gained by bacteria on treatment with antibiotics. In that respect, LexA is considered to be a potential drug target in Mycobacterium tuberculosis.
Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions and reported in the thesis, provide insights into hitherto underappreciated details of molecular structure and plasticity (Chapter 2). In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the ‘switch’ residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. Details of the interactions of ligands with the protein, characterised in the structures, could be useful for design of inhibitors against M. tuberculosis RecA.
Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from M. tuberculosis, M. smegmatis and E. coli and their ATP complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural frame work within which RecA molecules from different eubacteria function (Chapter 3). Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.
As described in Chapter 4, full length LexA, the N-terminal and C-terminal segments defined by the cleavage site, two point mutants involving changes in active site residues (S160A and K197A) and another involving change at the cleavage site (G126D) were cloned, expressed and purified. The wild type protein cleaves at basic pH. The mutants do not autocleave at basic pH even after incubation for 12 hours. The wild type and the mutant protein dimerise and bind DNA with equal facility. The C-terminal segment also dimerises, but has a tendency to form tetramer as well. The full length proteins including the mutants and the C-terminal segment crystallised. The structure of the crystals obtained for mutant G126D could not be solved. Each of the other crystals, four in number, contained only the catalytic core and a few residues preceding it, indicating that the full length proteins underwent cleavage, at the canonical cleavage site or elsewhere, during the long period involved in the formation of the crystals. Crystals obtained from the solutions of the wild type protein and the C-terminal segment contains dimers of the catalytic core. Crystals obtained using the active site mutants appear to contain different type of tetramers. One of them involves the swapping of the peptide segment preceding the catalytic core. Models of tetramerisation of the full length protein similar to those observed for the catalytic core are feasible. A model of a complex of MtLexA with M. tuberculosis SOS box could be readily built. In this complex, the mutual orientation of the two N-domains of the dimer is different from that in the EcLexA-DNA complex. | en_US |
