Regulation Mechanisms of the Dual ATPase in KaiC

KaiC is a dual ATPase, with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to ATP is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB. Significance Statement KaiC, a core clock protein in the cyanobacterial circadian clock system, hydrolyzes ATP at two distinct sites in a slow but ordered manner to measure the circadian time scale. We used biochemical and structural biology techniques to characterize the properties and interplay of dual ATPase active sites. Our results show that the N-terminal and C-terminal ATPases communicate with each other through an interface between the N-terminal and C-terminal domains in KaiC. The dual ATPase sites are regulated rhythmically in a concerted or opposing manner dependent on the phase of the circadian clock system, controlling the affinities of KaiC for other clock proteins, KaiA and KaiB. ### Competing Interest Statement The authors have declared no competing interest.