Biophysical insights into plasmid DNA binding by deinococcus grandis Dps nanocage

DNA-binding proteins from starved cells (Dps) are small multifunctional protein nanocages expressed by prokaryotes under oxidative stress or during starvation, acting as a key bacterial defense mechanism. Dps proteins protect DNA either through direct binding or by scavenging reactive oxygen species precursors. In most Dps homologs studied to date, DNA-binding is mediated by flexible, intrinsically disordered N- or C-terminal extensions. In this study, we investigated the interaction of Deinococcus grandis Dps (DgrDps) and a mutant variant, lacking the first 46 N-terminal residues, with supercoiled plasmid pUC19 using electrophoretic mobility shift assays (EMSA), DNase I protection assays, atomic force microscopy (AFM), and synchrotron radiation circular dichroism (SRCD). DgrDps binds supercoiled pUC19 with an apparent dissociation constant (KD) of 5.2 ± 0.3 µM, exhibiting positive cooperativity. Our results indicate that DNA binding is primarily mediated by the flexible N-terminal tails of DgrDps. AFM imaging revealed that DgrDps binds to multiple sites on the plasmid, inducing DNA bridging and compaction. Furthermore, the presence of 96 Fe2+/dodecamer increased the compaction of these protein-pUC19 complexes. This organization confers physical protection against DNase I digestion. Additionally, SRCD spectroscopy provided insights into the structural features and thermal stability of the DgrDps-DNA complexes.