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

Abstract

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.

Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock

“…We recently reported that KaiC alone undergoes phosphorylation in the absence of Mg 2+ in the reaction mixture. 11,16 Accordingly, ss-KaiC underwent phosphorylation in the absence of Mg 2+ (Figures 2C and S1C). Therefore, we concluded that there is no intrinsic mutantspecific property in ss-KaiC regarding the KaiC-alone phosphorylation and dephosphorylation pattern (Figure 2D).…”

“…Surprisingly, the freshly purified ss-KaiC in the reaction mixture did not show the oscillation, in contrast to wt-KaiC (Figures A and S2A). We reasoned that the freshly purified ss-KaiC did not undergo phosphorylation or dephosphorylation because it had already reached the equilibrium between the two antagonistic reactions . To shift the equilibrium, we dephosphorylated ss-KaiC by incubating it with KaiB at 30 °C overnight.…”

“…We reasoned that the freshly purified ss-KaiC did not undergo phosphorylation or dephosphorylation because it had already reached the equilibrium between the two antagonistic reactions. 16 To shift the equilibrium, we dephosphorylated ss-KaiC by incubating it with KaiB at 30 °C overnight. At time 0, KaiA was added into the reaction mixture containing dephosphorylated ss-KaiC.…”