| dc.description.abstract | In the global context, particularly in India, the two most important infectious diseases among many others are Tuberculosis (TB), an age-old disease and COVID-19, a new-age disease. This thesis focuses on some of the key proteins responsible for their pathogenesis.
In the second chapter, structural and functional characterization of Cystathionine-β-synthase from Mycobacterium tuberculosis (Mtb) was done using single-particle cryo-electron microscopy. CBS is the first enzyme of the transsulfuration pathway. This pathway produces hydrogen-di-sulfide (H2S) that scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), thereby protecting Mtb against oxidative stress. In this current study, recombinant MtbCBS protein was purified as a tetramer followed by several biochemical and biophysical studies to characterize oligomeric states, enzyme activities and allosteric activation of this enzyme. Native MtbCBS structure was resolved at a global resolution of 3.6 Å resolution calculated at 0.143 Fourier Shell Correlation (FSC). The local resolution showed the resolution of the core area between 3-3.2 Å, whereas the periphery area was resolved at a resolution of 3.4-3.6 Å. The atomic model of native MtbCBS was determined and demonstrated the domain boundaries. It is composed of N-terminal catalytic core region, C-terminal Bateman module and a 32 amino acid long linker between catalytic core and Bateman module. The active site is lysine 44, where pyridoxal phosphate (PLP) binds as a cofactor. Two Bateman module from two different monomers interacts antiparallelly during tetramerization. Site-directed mutagenesis was performed for the amino acids (L454, R450, S390, E388 and I357), present at the tetramer interface, followed by enzyme activities of mutated MtbCBS was measured. Current studies indicate that I357A mutation modulates the partial conversion (not the entire population) of tetramer to the dimer. Interestingly the basal specific activity of I357A mutant showed two-fold higher activity than native and no S-adenosyl methionine (SAM) activation, which is an allosteric activator of MtbCBS. Then, the cryo-EM model of MtbCBS in the presence of SAM (allosteric activator) was determined at a global resolution of 3.56 Å and built the atomic model. Conformational changes of the Bateman module (SAM binding area) were observed upon SAM interaction and visualized substrate channel opening due to allosteric activation of the protein. Last, MtbCBS structure was determined in the presence of substrate to understand the enzyme mechanism. To perform this, MtbCBS was incubated with serine to lock the enzyme at external aldimine and amino acrylate intermediate, and the structure was resolved at a global resolution of 4.25 Å. A significant conformational change was observed at the active site in the presence of serine. Lysine 44 – PLP interaction disappeared, and PLP-serine adduct formed in the presence of serine indicates the formation of external aldimine/amino acrylate. In future, these structural insights may enable us to develop specific inhibitors against the transsulfuration pathway to inhibit the growth of Mtb inside the host cell.
In the third chapter, the conformational flexibility of S protein was investigated at physiological pH (pH 7.4) and near physiological pH (pH 6.5 and pH 8.0) using single-particle cryo-EM. Many groups around the world reported structures of S protein (pre-fusion state) either at higher pH (pH 8.0) or lower pH (pH 4.5-5.5). These groups also found the variable proportion of open and closed conformations at different pH. S protein of SARS-CoV2 is highly susceptible to pH change converting open state to closed state and vice versa. The open conformation of S protein is a pre-requisite for SARS-CoV2 virus attachment with receptor hACE2 (Human-Angiotensin Converting Enzyme 2) and therefore beginning of the infection cycle. On the other hand, closed conformation will prohibit spike viral attachment to hACE2. The transition from the closed state to an open state is regulated by RBD (receptor binding domain) movement. In this chapter, various intermediate conformations of open and closed states of S protein were resolved (3.8-5.4 Å resolution), which suggests the flexible nature of RBD and NTD (N-terminal domain). Heterogeneity in both open and closed conformations was observed, or in other words, not all S proteins orient the same way. This heterogeneity at RBD and NTD generates a different solvent accessible surface area of different amino acids results in differential neutralising antibodies binding. Forty key interacting residues were chosen, which showed remarkable heterogeneity in solvent accessibility across different conformers over the pH range. The propensity of open and closed conformations was quantified over the selected pH range. A higher proportion of open state conformation (68%) at pH 7.4 was observed than pH 6.5 and 8.0. This explains why the virus successfully infects humans at physiological pH. Overall, the inherent structural flexibility of S protein at different pH was demonstrated.
In the fourth chapter, the overexpression of full-length PKS12 was optimized. PKS12, a type I polyketide synthase, is the largest open reading frame present in Mtb. Unlike other PKSs, PKS12 follows a different biosynthetic pathway called ‘modularly-iterative’ to produce polyketides. PKS12 is a multi-modular and multi-functional mega enzyme complex which assembles simple CoA thioesters to form a complex metabolite called MPM. PKS12 was purified in different states of oligomer (large supramolecular assembly) and dimer. The monomeric molecular weight of PKS12 is 431 kDa. The interesting phenomenon is PKS12 dimers undergoes a dramatic conformational change to spherical higher-order oligomer in the presence of extender unit malonyl CoA, methyl malonyl CoA and starting unit fatty acid; and visualized using negative staining TEM. It does so to facilitate the substrate transfer during enzymatic reaction. This study provides a unique oligomerisation mechanism of PKS12 in the presence of substrates, which provides a new perspective in the research field.
To summarise, this thesis provides insight into the structural and functional domain of the proteins responsible for the successful pathogenesis of Mycobacterium tuberculosis and SARS-CoV2. | en_US |
