Abstract text (incl. figure legends and references)

Protein nanocages have been explored as potential carriers in biomedicine. These caged structure could be engineered by loading therapeutic and diagnostic molecules in the core, while their external surfaces can be used to enhance their biocompatibility and targeting abilities.These cage-like proteins have propensity to self-assemble from simple identical building blocks to highly ordered architecture. DNA binding proteins under starvation (Dps),are one of such cage forming protein which form dodecamer with pore diameter of 5nm. It helps in storing the iron to avoid the formation of free radical. It also helps in binding and condensing the DNA to protects the DNA from various oxidative stressesunder starvation condition. These properties of Dps protein could be used to develop DNA-protein nanocage for drug delivery system.

One of such protein is M. smegmatis MsDps2, also showed DNA binding and iron storage capacity. MsDps2 binds the DNA without having any known DNA binding motifs. It is important to know the interaction of MsDps2 and DNA to explain the causes of bacterial resistance to antibiotics and to develop new antibacterial drug. Additionally, this DNA-protein interactions are important to know to construct the cage from both protein and DNA. However, different models were proposed for Dps and DNA interaction. But till date, there is a lag of structural information at atomic level for this Dps, including MsDps2 and DNA interaction

To decipher the MsDps2 and DNA interaction, we performed EMSA, MST, CD and Fluorometry biochemical experiments.Furthermore, we employed single-molecule imaging (TIRF), negative staining TEM and cryo-EM structural analysis to characterize the MsDps2-DNA interactions. All the above mentioned experiments were performed using different length and concentrations of MsDps2 protein

In this study, we reported that MsDps2 binds with DNA cooperatively and shows its compaction. EMSA studies suggested that the difference in the degree of compaction depends on DNA length and concentration of protein. We have used TIRF microscopy to further validate the compaction of DNA cooperatively on a real-time scale.Furthermore, our negative staining TEM imaging suggests that MsDps2 binds to DNA, which looks like pearls on a thread.

To decipher the structure of the MsDps2-DNA complex, we have performed cryo-EM with 600bp of DNA with MsDps2.We resolved the structure of the MsDps2-DNA complex at a resolution of 7Å, whereas the core MsDps2 was solved at 2.4Åresolution.Based on this structural information, we have mutated four arginine residues (R4E,R5E,R104E,R114E) which were involved in DNA binding. Mutated constructs showed less affinity to DNA binding; however, there is no effect on MsDps2 oligomerisation. Our cryo-EM based structural studies might help us to employ bioengineer the MsDps2 constructs to develop new protein-DNA cargo.We plan to integrate our work with other experimental and computational methods like Molecular dynamics simulations, SAXS and computational modelling (AlphaFold and Haddock) to get more insightsinto protein-DNA interaction and characterize its flexibility.