Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock.

Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC's two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported to be arrhythmic in vivo. However, we found that the mutant KaiC gradually loses the ability to run in an autonomous manner and stays constitutively phosphorylated after 3 cycles in vitro.

Shift in Conformational Equilibrium Underlies the Oscillatory Phosphoryl Transfer Reaction in the Circadian Clock.

Oscillatory phosphorylation/dephosphorylation can be commonly found in a biological system as a means of signal transduction though its pivotal presence in the workings of circadian clocks has drawn significant interest: for example in a significant portion of the physiology of Synechococcus elongatus PCC 7942. The biological oscillatory reaction in the cyanobacterial circadian clock can be visualized through its reconstitution in a test tube by mixing three proteins-KaiA, KaiB and KaiC-with adenosine triphosphate and magnesium ions. Surprisingly, the oscillatory phosphorylation/dephosphorylation of the hexameric KaiC takes place spontaneously and almost indefinitely in a test tube as long as ATP is present. This autonomous post-translational modification is tightly regulated by the conformational change of the C-terminal peptide of KaiC called the "A-loop" between the exposed and the buried states, a process induced by the time-course binding events of KaiA and KaiB to KaiC. There are three putative hydrogen-bond forming residues of the A-loop that are important for stabilizing its buried conformation. Substituting the residues with alanine enabled us to observe KaiB's role in dephosphorylating hyperphosphorylated KaiC, independent of KaiA's effect. We found a novel role of KaiB that its binding to KaiC induces the A-loop toward its buried conformation, which in turn activates the autodephosphorylation of KaiC. In addition to its traditional role of sequestering KaiA, KaiB's binding contributes to the robustness of cyclic KaiC phosphorylation by inhibiting it during the dephosphorylation phase, effectively shifting the equilibrium toward the correct phase of the clock.

The Circadian Clock-A Molecular Tool for Survival in Cyanobacteria.

Cyanobacteria are photosynthetic organisms that are known to be responsible for oxygenating Earth's early atmosphere. Having evolved to ensure optimal survival in the periodic light/dark cycle on this planet, their genetic codes are packed with various tools, including a sophisticated biological timekeeping system. Among the cyanobacteria is Synechococcus elongatus PCC 7942, the simplest clock-harboring organism with a powerful genetic tool that enabled the identification of its intricate timekeeping mechanism. The three central oscillator proteins-KaiA, KaiB, and KaiC-drive the 24 h cyclic gene expression rhythm of cyanobacteria, and the "ticking" of the oscillator can be reconstituted inside a test tube just by mixing the three recombinant proteins with ATP and Mg2+. Along with its biochemical resilience, the post-translational rhythm of the oscillation can be reset through sensing oxidized quinone, a metabolite that becomes abundant at the onset of darkness. In addition, the output components pick up the information from the central oscillator, tuning the physiological and behavioral patterns and enabling the organism to better cope with the cyclic environmental conditions. In this review, we highlight our understanding of the cyanobacterial circadian clock and discuss how it functions as a molecular chronometer that readies the host for predictable changes in its surroundings.

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock.

The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation. Among the clock proteins, the pseudo-receiver domain of KaiA and CikA can sense external cues by detecting the oxidation state of quinone, a metabolite that reflects the light/dark cycle, although the molecular mechanism is not fully understood. Here, we show the antagonistic phase shifts produced by the quinone sensing of KaiA and CikA. We introduced a new cyanobacterial circadian clock mixture that includes an input component in vitro. KaiA and CikA cause phase advances and delays, respectively, in this circadian clock mixture in response to the quinone signal. In the entrainment process, oxidized quinone modulates the functions of KaiA and CikA, which dominate alternatively at day and night in the cell. This in turn changes the phosphorylation state of KaiC-the central oscillator in cyanobacteria-ensuring full synchronization of the circadian clock. Moreover, we reemphasize the mechanistic input functionality of CikA, contrary to other reports that focus only on its output action.

CikA Modulates the Effect of KaiA on the Period of the Circadian Oscillation in KaiC Phosphorylation.

Cyanobacteria contain a circadian oscillator that can be reconstituted in vitro. In the reconstituted circadian oscillator, the phosphorylation state of KaiC oscillates with a circadian period, spending about 12 h in the phosphorylation phase and another 12 h in the dephosphorylation phase. Although some entrainment studies have been performed using the reconstituted oscillator, they were insufficient to fully explain entrainment mechanisms of the cyanobacterial circadian clock due to the lack of input pathway components in the in vitro oscillator reaction mixture. Here, we investigate how an input pathway component, CikA, affects the phosphorylation state of KaiC in vitro. In general, CikA affects the amplitude and period of the circadian oscillation of KaiC phosphorylation by competing with KaiA for the same binding site on KaiB. In the presence of CikA, KaiC switches from its dephosphorylation phase to its phosphorylation phase prematurely, due to an early release of KaiA from KaiB as a result of competitive binding between CikA and KaiA. This causes hyperphosphorylation of KaiC and lowers the amplitude of the circadian oscillation. The period of the KaiC phosphorylation oscillation is shortened by adding increased amounts of CikA. A constant period can be maintained as CikA is increased by proportionally decreasing the amount of KaiA. Our findings give insight into how to reconstitute the cyanobacterial circadian clock in vitro by the addition of an input pathway component, and explain how this affects circadian oscillations by directly interacting with the oscillator components.

Magnesium Regulates the Circadian Oscillator in Cyanobacteria.

The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesium-based hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established.