Reversible autoinhibition of the bacterial replication-licensing Hda-β clamp complex

Bacterial DNA replication must be tightly controlled to ensure once-per-cell-cycle initiation, requiring precise regulation of the initiator DnaA. The conserved RIDA pathway inactivates DnaA through the Hda-β sliding clamp (DnaN) complex, yet how this system is dynamically regulated has remained unclear. Here, we show that the Hda-DnaN complex exists as a concentration-dependent oligomeric ensemble rather than a static assembly. Using cryo-electron microscopy and mass photometry, we resolve multiple structural states, including an autoinhibited hetero-octamer, a hetero-hexamer intermediate, and a RIDA-compatible hetero-trimer. Structural comparison of these states suggests a plausible route of oligomeric interconversion involving progressive destabilization of Hda self-association, accompanied by exposure of the catalytic arginine finger required for DnaA inactivation. We further propose that this equilibrium operates as a titration-like mechanism that converts local protein concentration into functional states. Together, our findings establish reversible oligomerization as a potential regulatory principle for bacterial replication licensing.