Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited.

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.

Synthesis, crystal structures and docking studies of 2,7-diphenyl-1,4-diazepan-5-one derivatives.

BACKGROUND: 1,4-Diazepine derivatives are the seven membered, nitrogen containing heterocyclic ring systems possessing a wide range of therapeutic applications. 1,4-Diazepines attracted the attention of chemists and druggists due to their biological and medicinal properties, such as antimicrobial, anti-HIV and anticancer activities. Herein, we report the preparation, crystal structure determined by X-ray crystallographic methods and docking of the molecules with the potential target protein NS5B RNA polymerase. RESULTS: The crystal structures and conformational studies of 1,4-diazepine [t-3, t-6-dimethyl-r-2,c-7-diphenyl-1,4-diazepan-5-one(DIAZ1)] and its nitroso derivative [t-3, t-6-dimethyl-1-nitroso-r-2,c-7-diphenyl-1,4-diazepan-5-one(DIAZ2)] are reported. The analyses of the molecules reveal that the seven membered diazepine ring systems adopt chair and boat conformations in compounds DIAZ1 & DIAZ2, respectively. In DIAZ2, the oxygen O2A is disordered over two positions with the refined occupancies of 0.792(7): 0.208(7) in the nitroso group. In both DIAZ1 & DIAZ2, the symmetry related molecules form a hetero/homo-dimer through N-H…O hydrogen bonds. CONCLUSION: In this study, the crystal structures of two new 1,4-diazepines, namely t-3, t-6-dimethyl-r-2,c-7-diphenyl-1,4-diazepan-5-one and t-3, t-6-dimethyl-1-nitroso-r-2,c-7-diphenyl-1,4-diazepan-5-one were synthesized and characterized by X-ray crystallographic methods. The docking studies show that the compounds inhibit at the active site of the target protein and can be utilized as potential drug molecules. In both the compounds, N-H…O hydrogen bonds lead to dimer formation. In DIAZ2, additionally a couple of C-H…O interactions are noted between the molecules. Graphical AbstractStructure and docking studies of 1,4-diazapine derivatives.