Ordered Phosphorylation Governs Oscillation of a Three-Protein Circadian Clock

TLDR

The cyanobacterial circadian oscillator can be reconstituted in vitro with KaiA, KaiB, and KaiC, and its total phosphorylation oscillates with a circadian period, though the mechanism sustaining this oscillation is unclear. We demonstrate that four ordered KaiC phosphoforms arise from intrinsic autokinase and autophosphatase rates modulated by KaiA, with one phosphoform inhibiting KaiA through KaiB to provide feedback that sustains oscillation, and a model constrained by data reproduces the period and dynamics.

Abstract

The simple circadian oscillator found in cyanobacteria can be reconstituted in vitro using three proteins—KaiA, KaiB, and KaiC. The total phosphorylation level of KaiC oscillates with a circadian period, but the mechanism underlying its sustained oscillation remains unclear. We have shown that four forms of KaiC differing in their phosphorylation state appear in an ordered pattern arising from the intrinsic autokinase and autophosphatase rates of KaiC and their modulation by KaiA. Kinetic and biochemical data indicate that one of these phosphoforms inhibits the activity of KaiA through interaction with KaiB, providing the crucial feedback that sustains oscillation. A mathematical model constrained by experimental data quantitatively reproduces the circadian period and the distinctive dynamics of the four phosphoforms.