Structural and functional characterization of protein(s) involved in mycobacterial DNA repair

Abstract

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB). The dreaded disease accounts for ~25% of all cases known in the world in India. The pathogen is successful as it is able to evade the host immune system and persist for long years. This also involves protecting itself against the host-responses. In this context DNA repair is of paramount importance for the survival of the bug. In the absence of a mismatch repair (MMR) pathway in the mycobacterium, the non-canonical MMR by EndoMS/ NucS or DNA Base Excision Repair (BER) pathway assumes importance for countering host-inflicted oxidative DNA damaging responses. The BER pathway involves multiple players that detect specific types of damage and conclude with repair of the damage. Our laboratory has been involved in studying the specific and delicate interplay of protein-protein interactions in mycobacterial pre-replicative BER and their implications to the repair outcome. Understanding the underlying molecular mechanisms is expected to suggest alternate therapeutic strategies. Indeed, components of the pathway like the NAD+-dependent DNA ligase (MtbLigA), Sliding DNA ß-clamp, Class II AP-endonuclease, glycosylases and other interacting factors have been shown to be important for the survival of the pathogen and also critical players. In this context, our laboratory has identified several classes of inhibitors that can distinguish between human DNA Lig I and MtbLigA. We have also identified specific inhibitors that disrupt DNA ß-clamp –partner protein interactions, among others. We had also earlier reported the characterization of the DNA ß-clamp-XthA complex and showed that the mode of interactions is DNA substrate dependent. We set out to identify, characterize and functionally understand the roles of other BER complexes (BERosomes), the underlying molecular mechanisms and inhibitors that could disrupt the complexes for further development as a therapeutic strategy. We have carried out the study using a combination of structural biology tools like X-ray crystallography, Small-Angle X-ray scattering (SAXS), Surface plasmon resonance, Isothermal calorimetry, specifically designed in vitro and in vivo assays, and computational biology approaches. The present work is focused on the structural characterization of NAD+-dependent DNA ligase with the series of crystal and solution structures of adenylation reaction intermediates that explains the cofactor-induced serial-remodeling of the active site. This also involved identifying small-molecule fragment inhibitors and demonstrating their mode of interactions with LigA through co-crystal structures. We also report the structural characterization of an intriguing MtbLigA-MtbXthA complex. While v XthA is an initial player in the BER pathway, LigA is the terminal enzyme that completes the repair. Among other results, we have identified a novel fundamental interaction of the bacterial BER pathway that is apparently conserved. The studies suggest a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER. The thesis is divided in to seven sections that briefly described below- Chapter 1 This chapter acquaints the reader with the different kinds of DNA damage and the repair mechanisms. The focus is on base excision repair pathway (BER) and the BERosomes formed at the damaged site. This chapter also summarizes the potential of BER pathway and protein-protein interactions at the damaged site as a target for new therapeutic treatment. Chapter 2 Here, the various techniques and experimental approaches used to clone and purify recombinant proteins/ mutants reported in the present study are detailed. It also covers the various biochemical and biophysical assays used to characterize the respective protein-protein interactions. This chapter details the methods used to solve the crystal and solution structures of NAD+-dependent DNA ligase wild type and mutants. It also covers the experimental approaches used to identify and evaluate specific inhibitors of MtbLigA and MtbXthA. Chapter 3 reports the results regarding the mechanism of cofactor-induced serial remodeling of MtbLigA with the help of crystal and solution structures of various reaction intermediates. Also presented, is the mechanism of NMN release from the active site of MtbLigA in order to prevent the reaction reversal. The chapter also reports the details of conformational changes of MtbLigA in the presence of nicked DNA substrate with the help of SAXS studies and the link to the overall ligase reaction. Chapter 4 reports the determination/ identification of the residues that play a crucial role in the relative movements of Ia and 1b subdomains of the MtbLigA adenylation domain and also the conserved residues of BRCT domain. Structure guided mutational analysis of residues from the adenylation and BRCT domains respectively has identified novel roles during DNA ligation reaction. Chapter 5 details the structural characterization of the MtbLigA-MtbXthA complex. With the help of various techniques including SAXS, SPR, SEC, ITC and FRET we determined the motifs and residues on both MtbLigA and MtbXthA responsible for interaction. We also rationalized why the initial player (XthA) of the BER pathway interacts with the final vi component (LigA) and suggest that this is a conserved BER interaction that is needed to prevent futile cleavage and ligation cycles. Chapter 6 reports the evaluation of small molecule fragments from the commercially procured BioNet fragment library as potential and specific inhibitors of the MtbLigA. The chapter gives the details of the in vitro and in vivo antibacterial assays used to test their inhibition potency. The studies including co-crystallization of small molecule fragments with the adenylation (AdD) domain have led to the identification of small-molecule fragments that competitively bind to the NMN binding site. Chapter 7 is the final chapter and reports the identification of small molecule fragments from the BioNet library and Medicines for Malaria initiative MMV pathogen box as potential and specific inhibitors of the MtbXthA. The chapter gives the details of the in vitro and in vivo antibacterial assays used to test their inhibition potency. The X-ray crystallography data and Small Angle X-ray scattering data reported in the study have been submitted to the Protein Data Bank (PDB) and Small Angle Scattering Biological Data Bank (SASBDB) respectively and are listed below