Subunit shuffling dynamics in KaiC's central hub reveal the synchronization mechanism of the cyanobacterial circadian clock

Protein complexes are critical for cellular functions, and subunit exchange within these complexes is increasingly recognized as a key regulatory mechanism. In the cyanobacterial circadian clock, subunits shuffling of the core clock protein KaiC is thought to synchronize the clock, though the underlying mechanism remains unclear. We developed a chromatography-based method to monitor the shuffling dynamics of hexamerization domain of KaiC (KaiC-CI) and found that ATPase activity is essential for this process. By analyzing experiment data with quantitative models, we found that KaiC-CI hexamer stochastically disassembles into two oligomers for shuffling after hydrolysis. Further, by assuming a hidden conformation for post-hydrolysis hexamers, we established an ATPase activity-dependent model that quantitatively describes the shuffling dynamics of KaiC-CI hexamers, linking the shuffling rate to ATP hydrolysis and nucleotide exchange rates. Using this model, we estimated the shuffling dynamics of full-length KaiC with indirect experimental data. Our findings suggest that KaiCs phosphorylation states regulate nucleotide exchange rates in the CI domain, thereby modulating ATPase activity and influencing subunit shuffling. This study provides a mechanistic framework for understanding the role of ATPase activity in subunit exchange and its implications for circadian clock regulation.