Circadian clocks generate slow and ordered cellular dynamics but consist of fast-moving bio-macromolecules; consequently, the origins of the overall slowness remain unclear. We identified the adenosine triphosphate (ATP) catalytic region [adenosine triphosphatase (ATPase)] in the amino-terminal half of the clock protein KaiC as the minimal pacemaker that controls the in vivo frequency of the cyanobacterial clock. Crystal structures of the ATPase revealed that the slowness of this ATPase arises from sequestration of a lytic water molecule in an unfavorable position and coupling of ATP hydrolysis to a peptide isomerization with high activation energy. The slow ATPase is coupled with another ATPase catalyzing autodephosphorylation in the carboxyl-terminal half of KaiC, yielding the circadian response frequency of intermolecular interactions with other clock-related proteins that influences the transcription and translation cycle.

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring. During the phosphorylation cycle, the C-terminal domains of KaiC are repositioned in a stepwise manner to affect global expansion and contraction motions of the C-terminal ring. Arg393 of KaiC has a critical function in expanding the C-terminal ring and its replacement with Cys affects the temperature compensation of the period-a fundamental property of circadian clocks. The conformational ticking of KaiC observed here in solution serves as a timing cue for assembly/disassembly of other clock proteins (KaiA and KaiB), and is interlocked with its auto-inhibitory ATPase underlying circadian periodicity of cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser 431 and Thr 432 . The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser 431 -dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5′-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser 431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.