The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a selfsustained ∼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.dynamic allostery | NMR | protein structure | kinase | kinetics

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: CII ST flexible → CII SpT flexible → CII pSpT rigid → CII pST very-rigid → CII ST flexible . 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.

The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.

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