Structural and Functional Study on MazEF proteins from Mycobacterium tuberculosis

Abstract

The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the MazF toxin, which is ribonucleolytic enzyme and its cognate MazE antitoxin, which neutralizes the toxicity of MazF. MazF has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (EDF). Although it is well established that the increase in EDF-mediated toxicity of MazF drives a cell-killing phenomenon, the molecular details are poorly understood. Moreover, the divergence in sequences among reported EDFs suggests that each bacterial species has a unique EDF. To address these open questions, we report here the structures of MazF4 and MazEF4 complexes from M. tuberculosis, representing the first MazEF structures from this organism. We found that MazF4 possesses a negatively charged MazE4-binding pocket in contrast to the positively charged MazE-binding pockets in homologous MazEF complex structures from other bacteria. Moreover, using NMR spectroscopy and biochemical assays, we unraveled the molecular interactions of MazF4 with its RNA substrate and with a new EDF homolog originating from M. tuberculosis. The EDF homolog discovered here possesses a positively charged residue at the C-terminus, making this EDF distinct from previously reported EDFs. Overall, our results suggest that M. tuberculosis evolved a unique MazF and EDF and that the distinctive EDF sequence could serve as a starting point for designing new anti-tuberculosis drugs. We therefore conclude that this study might contribute to the development of a new line of anti-tuberculosis agents. MazE consists of DNA binding N-terminus and MazF binding C-terminus. MazE negatively regulates transcription of itself by binding to its operator as well as that of MazF. Compared to extensive studies conducted on MazF, there are only three structures of MazE available up to date. The three structures present MazE structure which is in complex with MazF but not on its own. In contrast to the structural similarity among C-terminus of MazE proteins, the structures of N-terminus are largely different. Therefore, we focused on the DNA binding domain of MazE in this study to understand the molecular detail of MazE-DNA interactions. Secondary structure prediction based on the primary structure of MazE2 from M. tuberculosis strongly suggests that the N-terminus of MazE2 forms Ribbon-Helix-Helix (R-H-H) motif, which is a typical DNA binding motif. Based on the secondary structure prediction, we obtained the stable MazE2 N-terminal domain which crystallized for 3D structure determination. The 3D structure indeed shows R-H-H motif. We also confirmed that this domain in itself can bind to its operator. Our NMR titration result reveals the MazE2 residues involved in DNA binding. Overall results show that N-terminus of MazE2 in itself can bind to its operator.