Biochemical basis of a 24-hour clock: Circadian clocks keep organisms in synch with such daily cycles as illumination, activity, and food availability. The circadian clock in cyanobacteria has the necessary 24-hour period despite its three component proteins having biochemical activities that occur on a much faster time scale. Abe et al. focused on the cyanobacterial clock component KaiC, an adenosine triphosphatase (ATPase) that can autophosphorylate and autodephosphorylate. The slow ATPase activity of KaiC, which is linked to a peptide isomerisation, provided the slow kinetics that set the speed of the 24-hour clock. Chang et al. found that another clock component, KaiB, also has slow changes in its protein conformation that help to set the oscillation period of the clock and its signaling output. Science , this issue pp. 312 and 324

Molecular clockwork from cyanobacteria: The cyanobacterial circadian clock oscillator can be reconstituted in a test tube from just three proteins—KaiA, KaiB, and KaiC—and adenosine triphosphate (ATP). Tseng et al. studied crystal and nuclear magnetic resonance structures of complexes of the oscillator proteins and their signaling output proteins and tested the in vivo effects of structure-based mutants. Large conformational changes in KaiB and ATP hydrolysis by KaiC are coordinated with binding to output protein, which couples signaling and the day-night transitions of the clock. Snijder et al. provide complementary analysis of the oscillator proteins by mass spectrometry and cryo–electron microscopy. Their results help to explain the structural basis for the dynamic assembly of the oscillator complexes. Science , this issue p. 1174 , p. 1181

The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a self-sustained ∼24-h rhythm of phosphorylation of KaiC. The mechanism propelling this oscillator has remained elusive, however. We show that stacking interactions between the CI and CII rings of KaiC drive the transition from the phosphorylation-specific KaiC–KaiA interaction to the dephosphorylation-specific KaiC–KaiB interaction. We have identified the KaiB-binding site, which is on the CI domain. This site is hidden when CI domains are associated as a hexameric ring. However, stacking of the CI and CII rings exposes the KaiB-binding site. Because the clock output protein SasA also binds to CI and competes with KaiB for binding, ring stacking likely regulates clock output. We demonstrate that ADP can expose the KaiB-binding site in the absence of ring stacking, providing an explanation for how it can reset the clock.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which, together with ATP, can generate a self-sustained ≈24 h oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate, whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the “A-loops”, are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. A dynamic equilibrium likely exists between the buried and exposed states, which determines the steady-state level of phosphorylation of KaiC. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state, KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation, and we show evidence in support of how this movement may be accomplished.

In the cyanobacterial circadian oscillator, KaiA and KaiB alternately stimulate autophosphorylation and autodephosphorylation of KaiC with a periodicity of approximately 24 h. KaiA activates autophosphorylation by selectively capturing the A loops of KaiC in their exposed positions. The A loops and sites of phosphorylation, residues S431 and T432, are located in the CII ring of KaiC. We find that the flexibility of the CII ring governs the rhythm of KaiC autophosphorylation and autodephosphorylation and is an example of dynamics-driven protein allostery. KaiA-induced autophosphorylation requires flexibility of the CII ring. In contrast, rigidity is required for KaiC-KaiB binding, which induces a conformational change in KaiB that enables it to sequester KaiA by binding to KaiA’s linker. Autophosphorylation of the S431 residues around the CII ring stabilizes the CII ring, making it rigid. In contrast, autophosphorylation of the T432 residues offsets phospho-S431-induced rigidity to some extent. In the presence of KaiA and KaiB, the dynamic states of the CII ring of KaiC executes the following circadian rhythm: . Apparently, these dynamic states govern the pattern of phosphorylation, ST → SpT → pSpT → pST → ST. CII-CI ring-on-ring stacking is observed when the CII ring is rigid, suggesting a mechanism through which the ATPase activity of the CI ring is rhythmically controlled. SasA, a circadian clock-output protein, binds to the CI ring. Thus, rhythmic ring stacking may also control clock-output pathways.

A biological clock in a test tube: The biological clock of cyanobacteria, which remarkably requires just three proteins, has been reconstituted in vitro in a system that allows detailed study of its inputs and outputs, bringing new understanding of how environmental signals can influence a biological oscillator and how the clock controls cellular events such as gene transcription. Chavan et al . extended the known in vitro function of the core clock components to include output signals to transcriptional regulation and allow monitoring through fluorescence measurements in real time. The authors combined crystallography, mutagenesis, and quantitative modeling to further explore the clock mechanism, which may enable future synthetic biology applications. —LBR