Mechanism of robust circadian oscillation of KaiC phosphorylation in vitro
📝 Abstract
By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, Kondo and his colleagues reconstituted the robust circadian rhythm of the phosphorylation level of KaiC (Science, 308; 414-415 (2005)). This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this paper, we discuss a model based on the two basic assumptions: The assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.
💡 Analysis
By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, Kondo and his colleagues reconstituted the robust circadian rhythm of the phosphorylation level of KaiC (Science, 308; 414-415 (2005)). This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this paper, we discuss a model based on the two basic assumptions: The assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.
📄 Content
1 Mechanism of robust circadian oscillation of KaiC phosphorylation in vitro
Kohei Eguchi*, Mitsumasa Yoda*, Tomoki P. Terada, and Masaki Sasai
Department of Computational Science and Engineering, Nagoya University, Nagoya 464-8603, Japan
ABSTRACT By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, Kondo and his colleagues reconstituted the robust circadian rhythm of the phosphorylation level of KaiC (Science, 308; 414-415 (2005)). This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this paper, we discuss a model based on the two basic assumptions: The assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.
*These two authors equally contributed to this work.
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INTRODUCTION
To resolve the mechanism of circadian rhythms, cyanobacteria have been studied as the
simplest organisms to exhibit rhythms. In a cyanobacterium Synechococcus elongatus
PCC 7942, the gene cluster kaiABC and their product proteins, KaiA, KaiB, and KaiC,
were shown to be essential in generating the rhythm (1), and intense interest has been
focused on these Kai proteins (2-19). In the recent study of Kondo and his colleague (20),
the circadian oscillation in the phosphorylation level of KaiC has been reconstituted in
vitro by incubating the mixture of KaiA, KaiB, and KaiC with ATP. This epoch-making
work indicates that protein-protein interactions (21) and the associated hydrolysis of ATP
(22) suffice to generate the rhythm in the absence of transcriptional or translational
processes.
Interactions between Kai proteins in vitro have been characterized in detail (4, 19, 21,
23-26): KaiC has the autophosphatase activity, so that KaiC is gradually
dephosphorylated when KaiC alone is incubated in vitro (19). KaiC is phosphorylated
when KaiC is incubated with KaiA (4, 19, 21), whereas KaiB attenuates the activity of
KaiA (4, 21). These observations suggest the scenario that KaiC with the low
phosphorylation level interacts with KaiA to make the phosphorylation level high, which
then allows interaction between KaiC and KaiB to make the phosphorylation level low.
The mixture solution of KaiA, KaiB, and KaiC, therefore should have two phases, the
phase of phosphorylation with lower affinity of KaiC to KaiB and the phase of
dephosphorylation with higher affinity of KaiC to KaiB. Such phosphorylation dependent
interactions among Kai proteins (21, 23) and existence of the two phases (23) were
confirmed by experiments. However, the precise mechanism of the robust oscillations
remains unclear, which calls for in silico modeling of the biochemical network of Kai
proteins.
Although the two phases of phosphorylation and dephosphorylation are discernible
experimentally, the difference of the system in these two phases is unknown. As KaiC
forms hexamer in solution (7, 8, 21), van Zon et al. (27) and Yoda et al. (28) have
assumed that a KaiC hexamer switches between two different structural states in the two
phases, each of which has the different affinity to KaiA or KaiB. Figure 1 is the simplest
reaction scheme based on this assumption of the allosteric transition of the KaiC hexamer.
In this reaction scheme, it is obvious that each individual KaiC hexamer switches
between the two phases and therefore shows oscillation in its phosphorylation level.
However, because the reactions occur stochastically at different timings, the
phosphorylation level of independent KaiC hexamers should be desynchronized, even if
all the KaiC hexamers are in the same oscillatory phase at the beginning. Such
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desynchronization should smear out the oscillation in the phosphorylation level of the ensemble of KaiC hexamers, but the observed clear and robust oscillation of the phosphryration level of the ensemble of many KaiC hexamers strongly suggests that there exists some communication among KaiC hexamers, which synchronizes the phosphorylation level of many KaiC hexamers. The simplest explanation of this communication would be the complex formation among KaiC hexamers to
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