Structural and mechanistic insights into the Rel protein of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is known to survive as slow-growing bacteria. However, under in-hostile conditions inside the human host, it can survive as persister. The persistent bacteria can stay dormant for months to years and hence doesn’t show any progression from latent infection to active disease development. During latent infection, Mtb is challenged by the host immune system in multiple ways which includes, hypoxia, acidification and nutrient starvation. To survive through these host responses, Mtb responds in an orchestrated manner. The elaborate machinery to withstand nutrient starvation enables the pathogen to survive, fatty acid limitation, carbon, iron and amino acid starvation. Amino acid starvation leads to stringent response which is understood in many bacteria including Mtb. However, the mechanism behind the regulation of stringent response independent to ribosomal complex have not been understood clearly. The stringent response is capable of changing gene expression at a global scale by means of stringent effector molecules which are a hyper- phosphorylated form of guanosine, ((p)ppGpp). The accumulation of (p)ppGpp is crucial to trigger stringent response which is regulated by proteins of the RelA/SpoT Homologous (RSH) family. Mtb shows a single functional copy of the relA gene which encodes the bifunctional protein MtRel, responsible to synthesize and hydrolyse the effector molecules. The MtRel is the only regulator of (p)ppGpp and can serve as a potential target for therapeutic interventions against drug tolerant tuberculosis and latent tuberculosis infection. In this thesis, the MtRel protein has been characterized structurally and mechanistically. The project provided the opportunity to study the structural domains of MtRel and thus insights were gained into the functional regulation of MtRel and its oligomerization state in solution. The catalytic N- terminal domains (NTDs), the hydrolase and the synthetase, are called as the MtRel NTD. The purified catalytically active recombinant MtRel NTD was found to be a dimer in solution as determined by SAXS studies. The atomic resolution structure was obtained by the X-ray crystallography agreed with the SAXS studies. The structure showed for the first time the arrangement of the mycobacterial hydrolase and synthetase domain in the absence of any substrate highlighting the unique features of binding sites in comparison to non-mycobacterial Rel proteins. SAXS studies in the presence of substrates further revealed unaltered overall shape of MtRel NTD in solution. The MtRel NTD, thus, showed the presence of specific pocket for synthetase domain and better resolution of the residues at the C-terminus of synthetase domain, providing new epitopes for drug discovery. The structure of the MtRel TGS determined by NMR spectroscopy and titration with deacylated tRNA answered the long- argued question about interaction of the TGS domain to deacylated tRNA in the absence of ribosomal machinery using biophysical methods. The titration-based mapping of residues involved in tRNA binding were identified and compared with the homologs of different species. The MtRel NTD in the presence of TGS domain showed almost similar synthetic activity as of MtRel NTD and approximately 3 fold higher than that of MtRel showing auto-inhibition by rest of the C-terminus MtRel segment. The MtRel ACT structure was determined as a dimer and the oligomeric state was further confirmed using SAXS. The crucial dimeric interface residues were identified, and the mutants were studied to understand the role of these residues in dimerization. The NMR spectroscopy titration experiments with branched chain amino acids revealed the specific binding of valine to the MtRel ACT and showed structural rearrangement upon valine binding. The studies unfolded the hypothetical mechanism of branched chain amino acids in allosterically regulating the catalytic activities of MtRel and hence acting as a sensor to determine the amino acid concentration. Overall, the work has guided in identifying species-specific epitopes in various domains and regulation by regulatory domains which can aid in structure aided drug discovery and therapeutic development.Doctor of Philosoph