Abstract:
DNA binding protein from starved cells (Dps) is a miniature ferritin complex, which plays a vital role in protecting bacterial DNA during starvation to maintain the integrity of bacteria under hostile conditions. Several approaches, including cryo-electron tomography, have been previously implemented by other research groups to decipher the structure of the Dps protein bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to identify the DNA binding residues. Like other bacteria, Mycobacterium smegmatis also expresses Dps2 (called MsDps2), which binds DNA to protect it under oxidative stress conditions. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps2 protein from Mycobacterium smegmatis. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA complexes and identified that the region close to the N-terminus confers the DNA binding property. Based on cryo-EM data, we also pinpointed several arginine residues, proximal to the DNA binding region, responsible for DNA binding. We also performed mutations of these residues, which dramatically reduced the MsDps2-DNA interaction. In addition, we proposed a model that elucidates the mechanism of DNA compaction, which adapts a lattice-like structure. We performed single-molecule imaging of MsDps2-DNA interactions that corroborate well with our structural studies. Taken together, our results delineate the specific MsDps2 residues that play an important role in DNA binding and compaction, providing new insights into Mycobacterial DNA compaction mechanisms under stress conditions.