Ordered Phosphorylation Governs Oscillation of a Three-Protein Circadian Clock

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 via 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.Circadian clocks coordinate metabolism and behavior with diurnal cycles in the environment (1). These clocks traditionally have been understood as transcriptional feedback oscillators in which clock genes repress their own synthesis, creating negative feedback that drives oscillation (1). However, pioneering work by Kondo and colleagues has shown that the circadian clock of the cyanobacterium Synechococcus elongatus requires neither transcription nor translation (2), and circadian oscillations can be reconstituted in vitro using only three proteins: KaiA, KaiB, and KaiC (3).KaiC is a hexameric enzyme (4) that can autophosphorylate (5) and autodephosphorylate (6) at both serine 431 (S431) and threonine 432 (T432) (7,8). The dimeric KaiA (9, 10) enhances the autophosphorylation of KaiC (11), whereas KaiB antagonizes the activity of . In the absence of KaiA, KaiC fully dephosphorylates (9). Complexes form between the Kai proteins (14), and the relative proportions of the KaiC-containing complexes oscillate (9, 15).The amount of phosphorylated KaiC oscillates with a circadian period (11). However, the total level of phosphorylation cannot be the only dynamical variable controlling the oscillator since it traverses the same value twice each day, but each time in a different † To whom correspondence should be addressed. E-mail: erin_oshea@harvard.edu. * These authors contributed equally to this work. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript direction (increasing during the subjective day and decreasing during the subjective night). Previous mathematical models have treated both phosphorylation sites as functionally equivalent (16)(17)(18)(19)(20)(21)(22)(23)(24), and have proposed additional dynamical variables arising from persistent conformational changes (18,(20)(21)(22)(23)(24) or long-lived heterocomplexes (16, 17); we hypothesized that additional variables could be found by examining the pattern of multisite phosphorylation of KaiC during the circadian cycle.We measured the time dependence of phosphorylation at S431 and T432 by SDS-PAGE (Fig. 1, A and B) and mass spectrometry (Fig. 1B), quantifying the four possible phosphorylation states: unphosphorylated (U-KaiC), p...

A genetic timer through noise-induced stabilization of an unstable state

“…In particular, this circuit topology is common among genetic oscillators, such as cell cycle and circadian clocks (30)(31)(32)(33)(34)(35). Additionally, transient cellular processes such as cell membrane polarization in neurons (36), yeast (37), and differentiation in bacteria (17,18,20) are also controlled by genetic circuits that are similar in architecture.…”

Period Robustness and Entrainability of the Kai System to Changing Nucleotide Concentrations

“…Both domains can bind and hydrolyze ATP, but only the CII domain can be phosphorylated. The CII domain has two phosphorylation sites, the threonine and the serine site, resulting in four different phosphorylation states for each monomer (31,34): unphosphorylated (U), phosphorylated only on serine (S), phosphorylated only on threonine (T), and double phosphorylated on both serine and threonine, (D).…”

“…Contrary to the Paijmans model and the Van Zon model, the Rust model describes the oscillations at the level of single monomers (26,27,31). As shown in Fig.…”

“…To this end, we measure the time between two important events during a full cycle of a hexamer, illustrated This paradox can be resolved by noting that the switch from the active to inactive state is determined by the difference between the number of phosphorylated serine and threonine sites, n S − n T , respectively. In the Paijmans model, the phosphotransfer rates for the threonine site are much faster than for the serine site (see (31,32,34)). Therefore, compared to the slow phosphorylation of the serine sites, n T will quickly reach its steady-state level during the phosphorylation phase.…”

“…al. (19); the extended monomer model, introduced by Phong and coworkers in (27) as an extension of the simpler model originally proposed in (31); and the model introduced in (32) which we refer to as the "Paijmans model" and which accounts for both KaiC its hexameric form and the fact that each KaiC monomer contains two distinct phosphorylation sites. In general, as shown in experiments, the effect of the ATP fraction on the oscillator is that, during the phosphorylation phase of the oscillation, the phosphorylation rate is roughly proportional to the ATP fraction, while during the dephosphorylation phase, the overall rate of dephosphorylation is unaffected (26).…”