Abstract EANA2026-248

Chroococcidiopsis is an extremophilic cyanobacterium considered a model organism for exploration astrobiology and the search for life beyond Earth due to its adaptations to harsh environments, including an outstanding tolerance to ionizing radiation. Key molecular determinants of this DNA damage resistance are the Dps (DNA-binding proteins from starved cells). These dodecameric, ferritin-like nucleoid-associated proteins protect DNA both chemically, by limiting the Fenton reaction via their ferroxidase activity, and physically, through direct DNA binding.

In Chroococcidiopsis sp. CCMEE 029, five dps paralogs (dps1-dps5) have been identified, but their structures and DNA-binding properties remain uncharacterised. This work presents a structural bioinformatics annotation of the five Dps and an ongoing computational assessment of their DNA-binding affinity, aimed at identifying which proteins, if highly expressed, would most effectively shield the DNA. 

Structural models of the five CCMEE 029 Dps proteins were generated using AlphaFold3 and inspected; Dps1, Dps2, Dps3 and Dps5 retain the BC helix, a structural feature that mediates inter-subunit contacts in canonical Dps, whereas Dps4 lacks it. Dps4 also lacks the ferritin-specific helix E and the bacterioferritin-characteristic methionine at position 52, suggesting it may represent an atypical, non-canonical Dps. A comparative sequence analysis against the characterised Dps proteins of Nostoc punctiforme and Anabaena sp. PCC 7120 was performed to predict and identify conserved functional features within the novel targets. Dps3 shares the highest identity with Alr3808, reported as the most efficient ferroxidase and DNA-binding Dps in Anabaena, and conserves its non-terminal lysine (K49), reported as essential for both activities. Sequence alignment together with structural superposition of the active site of the Dps3 model against its Nostoc ortholog NpDps4 further indicated that Dps3 carries a non-canonical “His-type” ferroxidase centre, in which two of the three carboxylate residues composing the centre are replaced by histidines.
Compared with the lysine-rich terminal tails classically responsible for Dps-DNA anchoring in several organisms, all five CCMEE 029 Dps exhibit a less pronounced terminal lysine enrichment.

Given these less positively charged tails, a pH- and electrostatics-dependent DNA-binding mode was hypothesised. For each of the five paralogs, a complex of two Dps dodecamers, the most common oligomeric state, bridging a double-stranded DNA molecule was assembled, reflecting the dense, aggregation-based binding mode typical of these assemblies.
The system was then prepared for molecular dynamics simulations; the Dps-DNA complexes were solvated, neutralised, energy-minimised, equilibrated and simulated in AMBER across a range of pH values, with protonation states assigned using H++.
These MD trajectories, and the following hydrogen-bonding, Buried Surface Area and MM-PBSA binding free-energy calculations, are still preliminary, but the effect of pH on the interaction is clearly significant.

These results provide a first, well-supported structural annotation of the Chroococcidiopsis sp. CCMEE 029 Dps family, including its putative dodecameric architecture, the ferroxidase centre morphology and conserved residues among the five Dps. Computational assessment of DNA-binding affinity is at an exploratory stage: the M12-6-4 force field, non-linear MM-PBSA, constant-pH MD, more accurate ionic strength conditions and reduced-oligomer models are being implemented to obtain more robust estimates.