Ito-Miwa, K.; Muranaka, T.; Kondo, T.; Terauchi, K.

Circadian clocks generate stable ~24-h rhythms with a defined period, temperature compensation, and entrainment to external cues that set phase. However, the molecular reactions that generate these features are not fully understood. In cyanobacteria, timekeeping is driven by the hexameric ATPase KaiC, which consists of two homologous domains, CI and CII, and whose enzymatic turnover underlies rhythmic phosphorylation. Here we identify ADP release as the rate-limiting step in the KaiC ATPase cycle and demonstrate that the KaiC ATPase nucleotide cycle integrates core properties of the circadian clock. Across KaiC period mutants, increased ADP occupancy is associated with longer periods. Elevated temperature shifts KaiC toward an ADP-bound state, offsetting the thermal acceleration of ATP hydrolysis. KaiB reinforces this ADP-bound conformation by inhibiting nucleotide exchange, thereby strengthening inhibition of CI ATPase activity of KaiC and tuning oscillation amplitude in a temperature-dependent manner. In contrast, KaiA accelerates ADP-to-ATP exchange within the KaiB-KaiC complex, thereby stimulating CI ATPase activity and promoting KaiB-KaiC dissociation prior to KaiC phosphorylation. Phosphorylation begins only after this transition, indicating that KaiC nucleotide-bound state sets the phase of the phosphorylation cycle. Collectively, these results establish the nucleotide cycle of KaiC ATPase as a unifying mechanism that connects molecular reactions to the defining properties of the cyanobacterial circadian clock.