Conformational Heterogeneity, Solution Structure and Autoregulatory Roles of the MazE9 Antitoxin from Mycobacterium tuberculosis

Abstract

The work presented in this thesis endeavors to decode the details of the dynamics of MazE9 antitoxin of the Mycobacterium tuberculosis MazEF9 toxin-antitoxin system and the transcriptional autoregulation effected by it. The MazEF9 TA system in Mycobacterium tuberculosis is implicated in persistence, intracellular survival and stress acclimatization. Regulation of this bicistronic operon at the level of transcription is a critical biochemical process that is key for the organism’s stress adaptation and intracellular survival. Here, we demonstrate that the MazE9 antitoxin presents flexible ‘Proline switches’ that rearrange the antitoxin in cis and trans states. We have identified 2 proline residues in the mobile C-terminal domain of MazE9 which undergo cis ⇌ trans isomerization, and hence contribute significantly to the conformational space available to MazE9 in the free energy landscape of the protein. A single site mutation of one of the participant prolines to an alanine locks the peptide backbone in the trans conformation, thereby restricting the exchange process. Additionally, we have reported the solution structure of the DNA binding domain of MazE9. 98.6 % of backbone torsion angles cluster in the most favored regions, and the remaining 1.4 % are found in the additionally allowed regions of the Ramachandran map, validating the excellent stereochemical quality of the calculated structure (PDB ID: 8IMH). Using a combination of biophysical and spectroscopic methods, we have scrutinized the thermodynamic and kinetic parameters operational in its interaction with the promoter/operator region, specific to the mazEF9 operon. The homodimeric N-terminal domain of MazE9 forms a tight complex with its operator DNA in an enthalpy-driven process with a dissociation constant of 0.53 uM at 1:1 stoichiometry. On the other hand, the thermodynamics and kinetics of the interaction of MazE9 with the functionally related mazEF6 operon, inferred from 2D lineshape analysis of NMR titration spectra, indicate that the potential for intracellular cross regulation is unlikely. A detailed biophysical and structural characterization of this antitoxin laid the groundwork for determination of the quaternary structure of the protein-DNA complex. A HADDOCK model of MazE9 bound to its operator DNA has been calculated based on the information on interacting residues obtained from these studies. The structural statistics of the water-refined models, including favorable interaction energies and a large buried surface area, attest to the good quality of the models generated. The structural and mechanistic insight into the interactions mediated by the MazE9 transcription factor may pave the way for structure guided drug design in the near future against this TA system involved in the virulence of Mycobacterium tuberculosis.