KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.     

Quantifying the Rhythm of KaiB-C Interaction for In Vitro Cyanobacterial Circadian Clock

An oscillator consisting of KaiA, KaiB, and KaiC proteins comprises the core of cyanobacterial circadian clock. While one key reaction in this process-KaiC phosphorylation-has been extensively investigated and modeled, other key processes, such as the interactions among Kai proteins, are not understood well. Specifically, different experimental techniques have yielded inconsistent views about Kai A, B, and C interactions. Here, we first propose a mathematical model of cyanobacterial circadian clock that explains the recently observed dynamics of the four phospho-states of KaiC as well as the interactions among the three Kai proteins. Simulations of the model show that the interaction between KaiB and KaiC oscillates with the same period as the phosphorylation of KaiC, but displays a phase delay of ～8 hr relative to the total phosphorylated KaiC. Secondly, this prediction on KaiB-C interaction are evaluated using a novel FRET (Fluorescence Resonance Energy Transfer)-based assay by tagging fluorescent proteins Cerulean and Venus to KaiC and KaiB, respectively, and reconstituting fluorescent protein-labeled in vitro clock. The data show that the KaiB∶KaiC interaction indeed oscillates with ～24 hr periodicity and ～8 hr phase delay relative to KaiC phosphorylation, consistent with model prediction. Moreover, it is noteworthy that our model indicates that the interlinked positive and negative feedback loops are the underlying mechanism for oscillation, with the serine phosphorylated-state (the "S-state") of KaiC being a hub for the feedback loops. Because the kinetics of the KaiB-C interaction faithfully follows that of the S-state, the FRET measurement may provide an important real-time probe in quantitative study of the cyanobacterial circadian clock.     

Effects of adenylates on the circadian interaction of KaiB with the KaiC complex in the reconstituted cyanobacterial Kai protein oscillator

Cyanobacteria are photosynthetic prokaryotes that possess circadian oscillators. Clock proteins, KaiA, KaiB, KaiC compose the central circadian oscillator, which can be reconstituted in vitro in the presence of ATP. KaiC has ATPase, autokinase, and autophosphatase enzymatic activities. These activities are modulated by protein–protein interactions among the Kai proteins. The interaction of KaiB with the KaiC complex shows a circadian rhythm in the reconstituted system. We previously developed a quantitative, real-time monitoring system for the dynamic behavior of the complex using fluorescence correlation spectroscopy. Here, we examined the effects of ATP and ADP on the rhythmic interaction of KaiB. We show that increased concentration of ATP or ADP shortened period length. Adding ADP to the Kai protein oscillation shifted its phase in a phase-dependent manner. These results provide insight into how circadian oscillation entrainment mechanism is linked to cellular metabolism.     

Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro

KaiA, KaiB, and KaiC are essential proteins of the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942. The phosphorylation cycle of KaiC that occurs in vitro after mixing the three proteins and ATP is thought to be the master oscillation governing the circadian system. We analyzed the temporal profile of complexes formed between the three Kai proteins. In the phosphorylation phase, KaiA actively and repeatedly associated with KaiC to promote KaiC phosphorylation. High levels of phosphorylation of KaiC induced the association of the KaiC hexamer with KaiB and inactivate KaiA to begin the dephosphorylation phase, which is closely linked to shuffling of the monomeric KaiC subunits among the hexamer. By reducing KaiC phosphorylation, KaiB dissociated from KaiC, reactivating KaiA. We also confirmed that a similar model can be applied in cyanobacterial cells. The molecular model proposed here provides mechanisms for circadian timing systems.     

Modelling circadian rhythms of protein KaiA, KaiB and KaiC interactions in cyanobacteria

Cyanobacteria are the simplest organisms known that exhibit circadian rhythms. The mechanism of circadian rhythm generation in cyanobacteria is different from eukaryotes. Based on the recent experiments about the interaction of KaiA, KaiB, and KaiC proteins with the generation of circadian rhythms in vitro, we developed a mathematical model to describe post-translational oscillations and the possible chemical reactions involved in the circadian clock mechanism of cyanobacteria. In this model, a series of differential equations, with linear kinetics for binding of proteins, Michaelis - Menten kinetics for enzymatic processes and a term including an explicit delay for dissociation of the KaiA/KaiB/phospho-KaiC complex, are proposed describing the dynamics of the chemistry. It is demonstrated that the mathematical system can lead to circadian oscillation within a range of parameter values.     

Circadian formation of clock protein complexes by KaiA,KaiB, KaiC,and SasA in cyanobacteria

Physical interactions among clock-related proteins KaiA, KaiB, KaiC, and SasA are proposed to be important for circadian function in the cyanobacterium Synechococcus elongatus PCC 7942. Here we show that the Kai proteins and SasA form heteromultimeric protein complexes dynamically in a circadian fashion. KaiC forms protein complexes of approximately 350 and 400-600 kDa during the subjective day and night, respectively, and serves as a core of the circadian protein complexes. This change in the size of the KaiC-containing complex is accompanied by nighttime-specific interaction of KaiA and KaiB with KaiC. In various arrhythmic mutants that lack each functional Kai protein or SasA, circadian rhythms in formation of the clock protein complex are abolished, and the size of the protein complexes is dramatically affected. Thus, circadian-regulated formation of the clock protein complexes is probably a critical process in the generation of circadian rhythm in cyanobacteria.     

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.     

Assembly and disassembly dynamics of the cyanobacterial periodosome

In vitro incubation of three Kai proteins, KaiA, KaiB, and KaiC, with ATP induces a KaiC phosphorylation cycle that is a potential circadian clock pacemaker in cyanobacterium Synechococcus elongatus PCC 7942. The Kai proteins assemble into large heteromultimeric complexes (periodosome) to effect a robust oscillation of KaiC phosphorylation. Here, we report real-time measurements of the assembly/disassembly dynamics of the Kai periodosome by using small-angle X-ray scattering and determination of the low-resolution shapes of the KaiA:KaiC and KaiB:KaiC complexes. Most previously identified period-affecting mutations could be mapped to the association interfaces of our complex models. Our results suggest that the assembly/disassembly processes are crucial for phase entrainment in the early synchronizing stage but are passively driven by the phosphorylation status of KaiC in the late oscillatory stage. The Kai periodosome is assembled in such a way that KaiA and KaiB are recruited to a C-terminal region of KaiC in a phosphorylation-dependent manner.     

蓝藻生物钟核心振荡器的KaiB-KaiC相互作用机制

蓝藻是已知的具有昼夜节律生物钟调控机制的最简单生物,其生物钟的核心是一个由三个蛋白质(Kai A、Kai B、Kai C)组成的,不依赖于转录翻译水平调控的核心振荡器.研究表明这三个蛋白质仅在体外试管中反应就会表现出周期性磷酸化振荡现象.分子水平研究表明:Kai A加速Kai C的自磷酸化,而Kai B抑制Kai A使Kai C去磷酸化,从而Kai C的磷酸化/去磷酸化形成周期性反复.但是Kai B如何与Kai A,Kai C相互作用,目前还不清楚.本文重点介绍了最近几年来在Kai B-Kai C相互作用机制上的研究进展,并结合我们的一些初步研究,对Kai B-Kai C相互作用的关键问题进行展望,以期为该体系的深入研究提供参考.     

Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB-KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.     

蓝藻生物钟分子机理的研究进展

蓝藻是具有内源性生物钟的简单生物.虽然蓝藻生物钟具有跟真核生物同样的基础特征,但其相关基因和蛋白质与真核生物没有同源性.蓝藻生物钟的核心是kai基因簇及其编码的蛋白KaiA,KaiB和KaiC.这三种Kai蛋白相互作用调节KaiC的磷酸化状态,从而产生昼夜节律信息.KaiC的磷酸化循环是昼夜节律的起博器,调控包括kai基因在内的相关基因的节律性表达.组氨酸蛋白激酶的磷酸化传递可将环境信息输入和将节律信息输出生物钟核心.     

The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi.     

Structure of the N-terminal domain of the circadian clock-associated histidine kinase SasA

Circadian oscillators are endogenous biological systems that generate the approximately 24 hour temporal pattern of biological processes and confer a reproductive fitness advantage to their hosts. The cyanobacterial clock is the simplest known and the only clock system for which structural information for core component proteins, in this case KaiA, KaiB and KaiC, is available. SasA, a clock-associated histidine kinase, is necessary for robustness of the circadian rhythm of gene expression and implicated in clock output. The N-terminal domain of SasA (N-SasA) interacts directly with KaiC and likely functions as the sensory domain controlling the SasA histidine kinase activity. N-SasA and KaiB share significant sequence similarity and, thus, it has been proposed that they would be structurally similar and may even compete for KaiC binding. Here, we report the NMR structure of N-SasA and show it to be different from that of KaiB. The structural comparisons provide no clear details to suggest competition of SasA and KaiB for KaiC binding. N-SasA adopts a canonical thioredoxin fold but lacks the catalytic cysteine residues. A patch of conserved, solvent-exposed residues is found near the canonical thioredoxin active site. We suggest that this surface is used by N-SasA for protein-protein interactions. Our analysis suggests that the structural differences between N-SasA and KaiB are the result of only a few critical amino acid substitutions.     

Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.     

Cooperative KaiA–KaiB–KaiC Interactions Affect KaiB/SasA Competition in the Circadian Clock of Cyanobacteria

The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~ 24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB–KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB–KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA–KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA–KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiB's favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.     

Cyanobacterial circadian clockwork: roles of KaiA,KaiB and the kaiBC promoter in regulating KaiC

Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not. One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter. On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo. The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA. Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC. These observations are integrated into a model proposing rhythmic changes in chromosomal status.     

Synchronization of Circadian Oscillation of Phosphorylation Level of KaiC In Vitro

In recent experimental reports, robust circadian oscillation of the phosphorylation level of KaiC has been reconstituted by incubating three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro. This reconstitution indicates that protein-protein interactions and the associated ATP hydrolysis suffice to generate the oscillation, and suggests that the rhythm arising from this protein-based system is the circadian clock pacemaker in cyanobacteria. The mechanism of this reconstituted oscillation, however, remains elusive. In this study, we extend our previous model of oscillation by explicitly taking two phosphorylation sites of KaiC into account and we apply the extended model to the problem of synchrony of two oscillatory samples mixed at different phases. The agreement between the simulated and observed data suggests that the combined mechanism of the allosteric transition of KaiC hexamers and the monomer shuffling between them plays a key role in synchronization among KaiC hexamers and hence underlies the population-level oscillation of the ensemble of Kai proteins. The predicted synchronization patterns in mixtures of unequal amounts of two samples provide further opportunities to experimentally check the validity of the proposed mechanism. This mechanism of synchronization should be important in vivo for the persistent oscillation when Kai proteins are synthesized at random timing in cyanobacterial cells.